Compositions of Microbiota and Methods Related Thereto

ABSTRACT

Methods and compositions are provided for treating weight related conditions and metabolic disorders by altering microbiota in a subject. One aspect provides methods and compositions to alter microbiota in a subject by administering to the subject a composition that includes a substantially purified microbiota from phyla such as Bacteroidetes, Proteobacteria, Firmicutes and Verrucomicrobia or orders such as Bacteroidales, Verrucomicrobiales, Clostridiales and Enterobacteriales or genera such as  Alistipes, Clostridium, Escherichia , and  Akkermansia . Another aspect includes a pharmaceutical composition for altering microbiota that includes a therapeutically effective amount of substantially purified microbiota and a pharmaceutically acceptable carrier. Yet another aspect includes methods for treating a disorder, such as obesity, in a subject in need of such treatment by changing relative abundance of microbiota in a gastrointestinal tract of the subject without or in addition to a surgical procedure.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of and claims the priority ofU.S. application Ser. No. 13/780,284 filed Feb. 28, 2013, which claimsthe benefit of U.S. Provisional Application Ser. No. 61/604,824 filedFeb. 29, 2012, entitled “Compositions of Microbiota and Methods RelatedThereto,” each of which is incorporated by reference herein in itsentirety.

FIELD OF THE INVENTION

The present invention relates to methods and compositions involvingmicrobiota for weight loss and the treatment of metabolic disease andits comorbidities, such as obesity, type II diabetes mellitus, etc.

BACKGROUND OF THE INVENTION

Obesity is a comorbidity of metabolic disease and represents the mostprevalent of body weight disorders, and it is the most importantnutritional disorder in the Western world, with estimates of itsprevalence ranging from 30% to 50% of the middle-aged population. Thenumber of overweight (defined as a person with a body mass index (BMI)equal to or greater than 25 kg/m²) and obese (defined as a person with aBMI equal to or greater than 30 kg/m²) Americans has continued toincrease since 1960, a trend that is not slowing down. Today,approximately 64.5% of adult Americans (about 199 million) arecategorized as being overweight or obese. Obesity is becoming a growingconcern as the number of people with obesity continues to increase andmore is learned about the negative health effects of obesity. Each year,obesity causes at least 300,000 deaths in the U.S., and healthcare costsof American adults with obesity amount to more than $147 billion(Centers for Disease Control and Prevention). Severe obesity, in which aperson has a BMI equal to or greater than 35, in particular posessignificant risks for severe health problems. Even mild obesityincreases the risk for premature death, diabetes, hypertension,atherosclerosis, gallbladder disease and certain types of cancer.Because of its high prevalence and significant health consequences, itstreatment should be a high public health priority. Reductions in weightas little as 5% of a patient's total body weight are associated withsignificant improvements in comorbidities associated with obesity andmetabolic disease such as type II diabetes mellitus, hypertension,hyperlipidemia, obstructive sleep apnea, gastroesophageal refluxdisease, breathing difficulties, etc. Accordingly, a great deal ofattention is being focused on treating patients with various stages ofobesity.

Surgical procedures to treat severe obesity and the associatedcomorbidities have included various forms of gastric and intestinalbypasses (stomach stapling), biliopancreatic diversion, adjustablegastric banding, vertical banded gastroplasty, gastric plications, andsleeve gastrectomies (removal of all or a portion of the stomach).Although these procedures can be performed using traditional opensurgical techniques, such surgical procedures have increasingly beenperformed laparoscopically. Reduced postoperative recovery time,markedly decreased post-operative pain and wound infection, and improvedcosmetic outcome are well established benefits of laparoscopic surgery,derived mainly from the ability of laparoscopic surgeons to perform anoperation utilizing smaller incisions of the body cavity wall. However,such surgical procedures risk a variety of complications during surgery,pose undesirable post-operative consequences such as pain and cosmeticscarring, and often require lengthy periods of patient recovery,particularly in the obese patient. Patients with obesity thus rarelyseek or accept surgical intervention, with less than about 1% ofpatients with obesity being surgically treated for this disorder.Furthermore, even if successfully performed and initial weight lossoccurs, surgical intervention to treat obesity may not result in lastingweight loss or improvements in comorbid conditions, thereby indicating apatient's need for additional, different, supplemental or complementaryobesity treatment(s).

Nonsurgical methods for treating obesity have also been developed.However, effective therapies for increasing energy expenditure leadingto improvements in metabolic outcomes, e.g., decreasing food intake,weight loss, glucose metabolism etc., have focused on pharmaceuticalapproaches, which have various technical and physiological limitations.

Accordingly, there remains a need for new methods and compositions forweight loss and treating metabolic disorders, such as obesity.

SUMMARY OF THE INVENTION

The present invention generally provides methods and compositions fortreating weight related conditions and disorders by altering microbiotain a subject. One aspect provides methods and compositions to altermicrobiota in a subject by administering to the subject a compositionthat includes a substantially purified microbiota from phyla such asBacteroidetes, Proteobacteria, Firmicutes and Verrucomicrobia or orderssuch as Bacteroidales, Enterobacteriales, Clostridiales, andVerrucomicrobiales, or genera such as Alistipes, Escherichia,Clostridium, or Akkermansia. Another aspect provides a pharmaceuticalcomposition for altering microbiota that includes a therapeuticallyeffective amount of substantially purified microbiota from phyla such asBacteroidetes, Proteobacteria, Firmicutes and Verrucomicrobia or orderssuch as Bacteroidales, Enterobacteriales, Clostridiales, andVerrucomicrobiales or genera such as Alistipes, Escherichia,Clostridium, or Akkermansia, and a pharmaceutically acceptable carrier.Methods for treating a disorder or condition associated with weight gainor methods for weight loss in a subject in need of such treatment bysubstantially increasing a relative abundance of microbiota from phylasuch as Bacteroidetes, Proteobacteria, Firmicutes and Verrucomicrobia ororders such as Bacteroidales, Enterobacteriales, Clostridiales, andVerrucomicrobiales or genera such as Alistipes, Escherichia,Clostridium, or Akkermansia in a gastrointestinal tract of the subjectwithout, or in addition to, a surgical procedure are also disclosed.

One aspect provides methods and compositions to alter microbiota byadministering one or more of a)-i) as follows: a) substantially purifiedVerrucomicrobia; b) substantially purified Enterobacteriales; c)substantially purified Bacteroidales; d) substantially purifiedClostridiales; e) substantially purified Alistipes; f) substantiallypurified Clostridium; g) substantially purified Akkermansia; h)substantially purified Escherichia; and i) a compound that whenadministered to a subject increases a relative abundance of at least oneof Verrucomicrobia, Verrucomicrobiales, Enterobacteriales,Bacteroidales, and Clostridiales in the subject and/or decreases arelative abundance of Tenericutes or Erysipelotrichales in the subject.In some embodiments, the methods and compositions can optionally includeadministering one or more of a) a compound that when administered to asubject alters a relative abundance of at least one of Bacteroidetes,Verrucomicrobia, Firmicutes, Tenericutes and Proteobacteria; b)substantially purified Bacteroidetes; c) substantially purifiedProteobacteria; d) substantially purified Firmicutes; and e)substantially purified Verrucomicrobia. The term “at least one of” asused throughout should be understood to include at least two of, atleast three of, at least four of, etc.

Another aspect provides methods and compositions to alter microbiota bydecreasing a relative abundance of Firmicutes or Tenericutes. In anexemplary embodiment, the relative abundance of Firmicutes orTenericutes in the subject can be decreased by at least about 5%. In aparticular embodiment, the relative abundance of eitherErysipelotrichales, Lactobacillus, or Allobaculum, or combinationsthereof, in the subject can be decreased by, for example, at least about5%.

A further aspect provides methods to alter microbiota in a subject byadministering to the subject a composition that includes a substantiallypurified Verrucomicrobia, a substantially purified Enterobacteriales, asubstantially purified Bacteroidales and/or a substantially purifiedClostridiales to treat a disorder selected from obesity, metabolicsyndrome, insulin deficiency, insulin-resistance related disorders,glucose intolerance, diabetes, non-alcoholic fatty liver, and abnormallipid metabolism is disclosed. The compositions can include at least oneof substantially purified Verrucomicrobia, a substantially purifiedEnterobacteriales, a substantially purified Bacteroidales and/or asubstantially purified Clostridiales that is a live bacterial strain.The methods can also include increasing a relative abundance of phylasuch as Bacteroidetes, Proteobacteria, Firmicutes and Verrucomicrobia ororders such as Bacteroidales, Enterobacteriales, Clostridiales, andVerrucomicrobiales or genera such as Alistipes, Escherichia,Clostridium, or Akkermansia in the subject (e.g. by at least about 5%).

The methods disclosed can further include delivering the composition toa target location within the subject. In one embodiment, the compositioncan be directly delivered to at least a stomach, a small intestine, anda large intestine of the subject. The composition can also be formulatedfor oral delivery.

The method can also include administering an osmotic laxative and/or anantibiotic to the subject and/or performing a surgical procedureselected from gastric bypass, duodenojejunal bypass, biliopancreaticdiversion, vertical sleeve gastrectomy, adjustable gastric banding,vertical banded gastroplasty, intragastric balloon therapy, gastricplication, Magenstrasse and Mill, small bowel transposition, biliarydiversion, duodenal endoluminal barrier, similar manipulations of thegastrointestinal tract, and other gastrointestinal bariatric andmetabolic procedures.

Another aspect includes a pharmaceutical or another composition foraltering microbiota that includes a therapeutically effective amount ofsubstantially purified microbiota from one or more phyla such asBacteroidetes, Proteobacteria, Firmicutes and Verrucomicrobia or orderssuch as Bacteroidales, Enterobacteriales, Clostridiales andVerrucomicrobiales or genera such as Alistipes, Escherichia,Clostridium, or Akkermansia and a pharmaceutically acceptable carrier.In other embodiments, a therapeutically effective amount of suchcompositions can be contained in food, drink, dietary supplement, and/orfood additive to be consumed by a subject.

Yet another aspect includes methods for treating obesity in a subject inneed of such treatment by substantially increasing a relative abundanceof at least one of Verrucomicrobiales, Enterobacteriales, Bacteroidales,and Clostridiales in a gastrointestinal tract of the subject without orin addition to a surgical procedure.

The methods can also include administering an additional agent, such asan antibiotic and/or an osmotic laxative, to the subject before,concurrent with, and/or after administration of the composition.

An additional aspect includes a kit for measuring the relative abundanceof Tenericutes, Mollicutes, Verrucomicrobiales, Enterobacteriales,Bacteroidales, and/or Clostridiales in a sample. The kit can include atleast a pair of primers that hybridize to Tenericutes, Mollicutes,Verrucomicrobiales, Enterobacteriales, Bacteroidales or Clostridialesnucleic acids and hybridization reagents.

A further aspect provides a method of altering microbiota in a subjectby administering a composition to the subject thereby alteringmicrobiota in the subject so that the microbiota mimics the microbiotafound in a subject responsive to a gastric bypass or othergastrointestinal bariatric or metabolic procedure.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be more fully understood from the following detaileddescription taken in conjunction with the accompanying drawings, inwhich:

FIG. 1 is a diagram of the gastrointestinal anatomy after Roux en-YGastric Bypass;

FIG. 2 is a diagram showing the procedure used to generate theexperimental animals. In particular, mice were separated into threegroups, animals that underwent surgical gastric bypass and maintained onhigh fat diet (RYGB), animals that underwent sham surgery and maintainedon high fat diet (SHAM), and animals that underwent sham surgery andwere calorically restricted to weigh the same as the RYGB animals, i.e.,weight matched (WMS);

FIG. 3A is a graph of the change in body weights over time of the RYGB,SHAM, and WMS animals after gastric bypass or sham operation as comparedto pre-operative weights;

FIG. 3B is a bar graph showing one week cumulative food intake of agroup of RYGB, SHAM, and WMS animals after gastric bypass or shamoperation;

FIG. 3C is a bar graph showing one week cumulative fecal energy outputof the RYGB, SHAM, and WMS animals from FIG. 3B;

FIG. 3D is a bar graph showing net energy intake of the RYGB, SHAM, andWMS animals from FIG. 3B over a one week period;

FIG. 3E is a graph of the change in oral glucose tolerance over time ofthe RYGB, SHAM, and WMS animals after gastric bypass or sham operation;

FIG. 3F is a graph of the change in insulin tolerance over time of theRYGB, SHAM, and WMS animals after gastric bypass or sham operation;

FIG. 4 is a bar graph showing the level of liver triglycerides in theRYGB, SHAM, and WMS animals measured at the time of tissue harvest;

FIG. 5 is a bar graph showing the level of serum triglycerides in theRYGB, SHAM, and WMS animals measured at the time of tissue harvest;

FIG. 6 is a bar graph showing the percentage of lean and fat weights ofRYGB, SHAM, and WMS mice as compared to total body weight at the time oftissue harvest;

FIG. 7 is a bar graph showing the percentage of adipose tissue found inthe epididymal and retroperitoneal fat pad of the RYGB, SHAM, and WMSanimals as compared to total body weight at the time of tissue harvest;

FIG. 8 is a flow diagram showing the analysis of microbiota in fecalsamples from from animals;

FIG. 9 is a graph showing the first principal coordinate analysis (PC1)from an Unweighted UniFrac-based analysis comparing fecal microbialcommunities of the animals;

FIG. 10 is a graph showing an unweighted UniFrac-based analysiscomparing microbial populations found in lumen and mucosa of the distalstomach remnant/gastric pouch (DS/GP), biliopancreatic limb (BP), Rouxlimb (Roux), common limb (CL), ileum, cecum, or colon of the animals;

FIG. 11A is a graph showing the relative abundance of Enterobacterialespopulations using phylogenetic information from fecal samples taken pre-and post-operative from the RYGB, SHAM, and WMS mice;

FIG. 11B is a graph showing the relative abundance of Enterobacterialespopulations over time of the RYGB, SHAM, and WMS animals after gastricbypass or sham operation as compared to pre-operative abundance;

FIG. 11C is a graph showing the mean fold-change of Enterobacterialescomparing RYGB to SHAM and WMS controls along the length of thegastrointestinal tract;

FIG. 12 is a bar graph showing the percentage relative abundance ofbacterial orders in RYGB, SHAM, and WMS mice in samples taken beforesurgery to 12 weeks after surgery, the upper bar highlights the increasein Enterobacteriales populations in RYGB animals and the lower barhighlights the increase in Verrucomicrobiales populations in RYGBanimals;

FIG. 13 is a bar graph showing the relative abundance of bacterialorders in RYGB, SHAM, and WMS mice throughout the gastrointestinaltract, the upper bar highlights the increase in Enterobacterialespopulations in RYGB animals and the lower bar highlights the increase inVerrucomicrobiales populations in RYGB animals;

FIG. 14 is a phylogenetic tree depicting nodes within the bacterialtaxonomic hierarchy that are significantly enriched in fecal samplesfrom RYGB (*), SHAM (**), and WMS (***) mouse fecal samples;

FIG. 15 shows a diagram of the procedure used to generate the recipientanimals. In particular, cecal content from RYGB, SHAM, and WMS mice wastransferred by oral gavage to germ-free (GF) recipient mice;

FIG. 16 is a graph of the percentage change in body weights over timeafter gavage treatment with samples as compared to pre-gavage treatmentweights: RYGB-R received cecal contents from RYGB mice, SHAM-R receivedcecal contents from SHAM mice, and germ-free controls were maintainedwithout exposure to any microbes;

FIG. 17 is a bar graph of the percentage change in body weight of therecipient animals 13 days after gavage treatment as compared topre-gavage treatment weights;

FIG. 18 is a bar graph showing the cumulative food intake of animalsgavage treated with samples from RYGB (RYGB-R), SHAM (SHAM-R), orcontrol (germ-free);

FIG. 19 is a bar graph showing the adiposity index of the recipientmice. In particular, decreased adiposity was transmissible via the gutmicrobiota from RYGB mice;

FIG. 20 is a bar graph showing leptin levels in recipient animals attime of tissue harvest;

FIG. 21 is a bar graph showing the level of serum triglycerides inrecipient animals measured at the time of tissue harvest;

FIG. 22A is a bar graph showing the level of energy expenditure ofanimals gavage-treated with samples from RYGB (RYGB-R), SHAM (SHAM-R),or control (GF) 5 days post-injection with doubly labeled water;

FIG. 22B is a bar graph showing the level of energy expenditure ofanimals gavage-treated with samples from RYGB (RYGB-R), SHAM (SHAM-R),or control (GF) 8 days post-injection with doubly labeled water;

FIG. 23A is a 72 hour time course graph of the respiratory quotientmeasured by indirect calorimetry of recipient animals 2 weeks followinggavage with cecal samples from RYGB (RYGB-R), SHAM (SHAM-R), or control(GF);

FIG. 23B is a graph showing the overall difference in respiratoryquotient between RYGB-R, SHAM-R, and GF groups from FIG. 24A;

FIG. 23C is the average difference in respiratory quotient during thelight cycles and the dark cycles among each recipient group;

FIG. 24A is a bar graph of the total cecal SCFAs of each donor group;

FIG. 24B is a series of graphs showing acetate, propionate, and butyrateas a percentage of total SCFAs in the donor groups;

FIG. 24C is a bar graph of the total cecal SCFAs of each recipientgroup;

FIG. 24D is a series of graphs showing acetate, propionate, and butyrateas a percentage of total SCFAs in the recipient groups; and

FIG. 25 is a bar graph showing the relative abundance of bacterialorders in recipient animals following gavage with samples of RYGB, SHAM,or WMS cecal content, the bar highlights the increase inVerrucomicrobiales populations in RYGB animals.

DETAILED DESCRIPTION OF THE INVENTION

Certain exemplary embodiments will now be described to provide anoverall understanding of the principles of the structure, function,manufacture, and use of the therapeutics and methods disclosed herein.One or more examples of these embodiments are illustrated in theaccompanying drawings. Those skilled in the art will understand that thetherapeutics and methods specifically described herein and illustratedin the accompanying drawings are non-limiting exemplary embodiments andthat the scope of the present invention is defined solely by the claims.The features illustrated or described in connection with one exemplaryembodiment may be combined with the features of other embodiments. Suchmodifications and variations are intended to be included within thescope of the present invention.

All publications, patents and patent applications cited herein arehereby incorporated by reference in their entirety. As used in thisspecification and the appended claims, the singular forms “a,” “an,” and“the” include plural references unless the content clearly dictatesotherwise. The terms used in this invention adhere to standarddefinitions generally accepted by those having ordinary skill in theart.

Studies show that the relationship between gut microbiota and humans isnot merely commensal (a non-harmful coexistence), but rather often is amutualistic, symbiotic relationship. Although animals can survive withno gut microbiota, the microorganisms perform a host of usefulfunctions, such as stimulating immune development, preventing invasionby pathogenic bacteria, regulating the development of the gut,fermenting unused dietary substrates, metabolism of glycans and aminoacids, synthesis of vitamins (such as biotin and vitamin K) andisoprenoids, biotransformation of xenobiotics, and directing the host tostore fats. It is therefore believed that changes in the composition ofthe gut microbiota could have important health effects.

Indeed, it has been discovered that a correlation between weight lossdue to surgical intervention, such as gastric bypass, and gut microbiotahas been observed. Therefore, the invention disclosed is generallydirected to therapeutic methods for weight loss and compositions fortreating disorders and conditions associated with weight gain, such asobesity, diabetes, and other metabolic disorders or comorbidities ofobesity, in a subject by altering the subject's gut microbiotapopulation, with the result of altering an energy balance in thesubject. Generally speaking, to increase energy utilization/expenditure,decrease body fat, or promote weight loss, the relative abundance ofmicrobiota within particular bacterial taxa can be altered. Alteringmicrobiota can include changing a relative abundance of microbiota byincreasing and/or decreasing the relative abundance of one or moremicrobiota.

The term “metabolic disorder” as used herein, refers to disorders,diseases, and conditions that are caused or characterized by abnormalweight gain, energy use or consumption, altered responses to ingested orendogenous nutrients, energy sources, hormones or other signalingmolecules within the body or altered metabolism of carbohydrates,lipids, proteins, nucleic acids or a combination thereof. A metabolicdisorder is associated with either a deficiency or excess in a metabolicpathway resulting in an imbalance in metabolism of nucleic acids,proteins, lipids, and/or carbohydrates. Factors affecting metabolisminclude, and are not limited to, the endocrine (hormonal) control system(e.g., the insulin pathway, the enteroendocrine hormones includingGLP-1, PYY or the like), the neural control system (e.g., GLP-1 or otherneurotransmitters or regulatory proteins in the brain) or the like. Somenon-limiting examples can be obesity, diabetes, including type IIdiabetes, insulin-deficiency, insulin-resistance, insulin-resistancerelated disorders, glucose intolerance, syndrome X, inflammatory andimmune disorders, osteoarthritis, dyslipidemia, metabolic syndrome,non-alcoholic fatty liver, abnormal lipid metabolism, cancer,neurodegenerative disorders, sleep apnea, hypertension, highcholesterol, atherogenic dyslipidemia, hyperlipidemic conditions such asatherosclerosis, hypercholesterolemia, and other coronary arterydiseases in mammals, and other disorders of metabolism.

Disorders also included are conditions that occur or cluster together,and increase the risk for heart disease, stroke, diabetes, and obesity.Having just one of these conditions such as increased blood pressure,elevated insulin levels, excess body fat around the waist or abnormalcholesterol levels can increase the risk of the above mentioneddiseases. In combination, the risk for coronary heart disease, stroke,insulin-resistance syndrome, and diabetes is even greater.

The increasing prevalence of obesity in the population has led to aparallel rise in surgical procedures, like bariatric surgery, as atreatment for obesity and related comorbid conditions. Surgicalprocedures can achieve a sustained weight reduction of up to 70% ofexcess body weight in the majority of patients, and are more effectivethan nonsurgical approaches. It has been discovered that gastric bypasscan not only lead to early satiety, satiation, increased energyexpenditure, improved glucose metabolism, and durable weight loss, butcan also alter the microbiota in the gastrointestinal tract of thesubject. Therefore, in an exemplary embodiment, a method of alteringmicrobiota in a subject can be used to treat a weight related disorder,such as obesity.

The terms “treating,” “treatment” or “intervention” refer to theadministration or delivery of one or more therapeutic agents,compositions or procedures to a subject who has a condition or disorderor a predisposition toward a condition or disorder, with the purpose toprevent, alleviate, relieve, alter, remedy, ameliorate, improve, affect,slow or stop the progression, slow or stop the worsening of the disease,at least one symptom of condition or disorder, or the predispositiontoward the condition or disorder.

The term “subject” as used herein refers to any living organism,including, but not limited to, humans, nonhuman primates such aschimpanzees and other apes and monkey species; farm animals such ascattle, sheep, pigs, goats and horses; domestic mammals such as dogs andcats; laboratory animals including rodents such as mice, rats, rabbitsand guinea pigs, and the like. The term does not denote a particular ageor sex. In a specific embodiment, the subject is human.

Microbiota

Methods and compositions are disclosed that include altering microbiotain a subject. As used herein, the term “microbiota” is used to refer toone or more bacterial communities that can be found or can exist(colonize) within a gastrointestinal tract of an organism. Whenreferring to more than one microbiota, the microbiota may be of the sametype (strain) or it may be a mixture of taxa, such as a mixture ofBacteroidetes, Firmicutes, Proteobacteria, Tenericutes, andVerrucomicrobia. In one aspect, methods and compositions are disclosedthat alter the relative abundance of microbiota from phyla such asBacteroidetes, Firmicutes, Tenericutes, Proteobacteria, andVerrucomicrobia or orders such as Bacteroidales, Erysipelotrichales,Clostridiales, Enterobacteriales and Verrucomicrobiales or genera suchas Alistipes, Clostridium, Escherichia, Allobaculum, or Akkermansia in agastrointestinal tract of a subject. The relative abundance microbiotacan be altered by administering a pharmaceutical composition thatincludes microbiota from phyla such as Bacteroidetes, Proteobacteria,Firmicutes and Verrucomicrobia or orders such as Bacteroidales,Enterobacteriales, Clostridiales and Verrucomicrobiales or genera suchas Alistipes, Escherichia, Clostridium, or Akkermansia or a compoundthat substantially increases the relative abundance of microbiota fromphyla such as Bacteroidetes, Proteobacteria, Firmicutes, andVerrucomicrobia or orders such as Bacteroidales, Enterobacteriales,Clostridiales and Verrucomicrobiales or genera such as Alistipes,Escherichia, Clostridium, or Akkermansia, or substantially decreases therelative abundance of microbiota from phyla such as Firmicutes orTenericutes or classes such as Mollicutes or orders such asErysipelotrichales or genera such as Allobaculum.

In another aspect, methods and compositions are disclosed thatselectively alter microbiota from phyla such as Bacteroidetes,Proteobacteria, Firmicutes, Tenericutes, and Verrucomicrobia or orderssuch as Bacteroidales, Enterobacteriales, Erysipelotrichales,Clostridiales, and Verrucomicrobiales or genera such as Alistipes,Escherichia, Clostridium, Akkermansia or Allobaculum in agastrointestinal tract of an organism. Bacteroidetes, Firmicutes,Proteobacteria, Tenericutes, Verrucomicrobia, Bacteroidales,Enterobacteriales, Erysipelotrichales, Clostridiales,Verrucomicrobiales, Alistipes, Escherichia, Clostridium, Akkermansia, orAllobaculum can be selectively altered by administering a food or foodsupplement that includes Bacteroidetes, Firmicutes, Proteobacteria,Verrucomicrobia, Bacteroidales, Enterobacteriales, Clostridiales,Verrucomicrobiales, Alistipes, Escherichia, Clostridium, and/orAkkermansia or can substantially increase or decrease the relativeabundance of Bacteroidetes, Firmicutes, Proteobacteria, Tenericutes,Verrucomicrobia, Bacteroidales, Enterobacteriales, Erysipelotrichales,Clostridiales, Verrucomicrobiales, Alistipes, Escherichia, Clostridium,Akkermansia, and/or Allobaculum in a gastrointestinal tract of anorganism.

Additionally, the methods may include altering bacterial taxa, such asaltering bacterial phyla, altering bacterial classes, bacterial orders,bacterial families, bacterial genera and/or bacterial species in agastrointestinal tract of an organism. In particular, five bacterialphyla are disclosed, Bacteroidetes, Firmicutes, Proteobacteria,Tenericutes, and Verrucomicrobia. The methods and compositions includealtering bacterial classes, bacterial orders, bacterial families,bacterial genera and/or bacterial species of one or more of thebacterial phyla Bacteroidetes, Firmicutes, Proteobacteria, Tenericutes,and Verrucomicrobia in a gastrointestinal tract of an organism. In anexemplary embodiment, the methods and compositions include alteringbacterial classes, bacterial orders, bacterial families, bacterialgenera and/or bacterial species of Bacteroidetes, Firmicutes,Proteobacteria, Tenericutes, or Verrucomicrobia in a gastrointestinaltract of an organism.

The phylum Verrucomicrobia is a newly described, divergent phylum withinthe Bacteria domain. Microbial communities of Verrucomicrobia have beenisolated from yeast, soil, feces and fresh and marine waters. Inaddition, extremely acidophilic members have been discovered from hotsprings that oxidize methane and use methane as a sole source of carbonand energy. Some species within Verrucomicrobia harbor genes homologousto those encoding eukaryotic tubulins. Akkermansia, found in thegastrointestinal tract and associated with gut health, can degrade mucinand host mucins as a sole source of carbon and nitrogen fuel. Atpresent, six monophyletic subdivisions (subphyla, classes) arerecognized within the phylum Verrucomicrobia on the basis of 16S rRNAgene library studies. In an exemplary embodiment, the composition canalter the relative abundance of bacteria from the classVerrucomicrobiae, bacteria from the order Verrucomicrobiales, bacteriafrom the family Verrucomicrobiaceae, and/or bacteria from the genusAkkermansia.

The phylum of Bacteroidetes contains three large classes of bacteria,Bacteroidia, Flavobacteria, and Sphingobacteria, that are distributed inthe environment, including in soil, in sediments, seawater and in theguts and on the skin of animals. The predominantly Gram-negativebacteria are the most well-studied phylum since they are commonly foundin the human intestine where they have a symbiotic host-bacterialrelationship with humans. They assist in breaking down food andproducing valuable nutrients and energy that the body needs. Bacteriafrom the genus Alistipes are generally bile acid resistant and mayincrease after a bile diversion-type surgery. In one embodiment, thecomposition can alter the relative abundance of bacteria from the classBacteroidia, bacteria from the order Bacteroidales, bacteria from thefamily Rikenellaceae, and/or bacteria from the genus Alistipes.

Proteobacteria is the largest phylum of bacteria. As a group, theseorganisms show extreme metabolic diversity and represent the majority ofknown bacteria of medical, industrial, and agricultural significance.This is an evolutionarily, geologically, and environmentally importantgroup. All Proteobacteria are Gram negative, with an outer membranecomposed of lipopolysaccharides. Many have gas vesicles, flagella, orcan move by gliding; they may have stalks, other appendages or theability to form multicellular fruiting bodies. Most members arefacultatively or obligately anaerobic, chemoautotrophs, andheterotrophic, but there are exceptions. Some species are able to carryout photosynthesis, others deposit sulphur within the cells or outside.

The Proteobacteria are divided into six sections, referred to as alphathrough zeta, based on rRNA sequences. The alpha, beta, delta, andepsilon sections are monophyletic. Gammaproteobacteria is paraphyleticwith respect to beta proteobacteria, i.e. Gammaproteobacteria consistsof almost all the descendants of Betaproteobacteria. In one embodiment,the composition can alter the relative abundance of bacteria from theclass Gammaproteobacteria, bacteria from the order Enterobacteriales,bacteria from the family Enterobacteriaceae, and/or bacteria from thegenus Escherichia.

Tenericutes are a phylum of bacteria that lack a cell wall and do notcontain muramic acid. This phylum includes the class, Mollicutes, thatcontains mycoplasms. Some are parasites of various animals and plants,living on or in the host's cells. Individual organisms are generallyvery small, typically only 0.2-0.3 μm in size and with a small genomesize. They vary in form, although most have sterols that make the cellmembrane somewhat more rigid. Many are able to move about throughgliding or twisting. In one embodiment, the composition can alter therelative abundance of bacteria from the class Mollicutes, and/orbacteria from the genus Allobaculum.

Firmicutes are a phylum of bacteria that are mostly Gram positive. Afew, however, have a porous pseudo-outer-membrane that causes them tostain Gram negative. Scientists once classified the Firmicutes toinclude all Gram-positive bacteria, but have recently defined them to beof a core group of related forms called the low −G+C. They arepredominantly round cells, called cocci (singular coccus), or rod-likeforms (bacillus). Many Firmicutes produce endospores, which areresistant to desiccation and can survive extreme conditions allowingthem to live in various environments. The group is typically dividedinto Clostridia, which are anaerobes, and Bacilli, which are obligate orfacultative anaerobes. The class Clostridia includes the familyLachnospiraceae, the family Ruminococcaceae, and the genus Clostridium.The class Bacilli includes the genus Lactobacillus. In one embodiment,the composition can alter the relative abundance of bacteria from theclass Erysipelotrichi, bacteria from the order Erysipelotrichales,and/or bacteria from the family Erysipelotrichaceae.

In an exemplary embodiment, the microbiota disclosed can be a probioticbacteria or non-pathogenic bacteria which, when the relative abundancealtered, can confer a health benefit to the host. Probiotic strainsgenerally have the ability to survive the passage through the upper partof the digestive tract when administered orally. They are nonpathogenic,non-toxic and exercise their beneficial effect on health on the onehand, possibly via ecological interactions with the resident flora inthe digestive tract, and on the other hand, possibly via their abilityto influence the immune and metabolic systems in a positive manner viathe “GALT” (gut-associated lymphoid tissue). Depending on the definitionof probiotics, these microbiota, when given in a sufficient number, havethe ability to progress live through the intestine; however, they do notcross the intestinal barrier in large numbers and their primary effectsare therefore induced in the lumen and/or the wall of thegastrointestinal tract. They then form part of the resident flora. Thiscolonization (or transient colonization) allows the probiotic microbiotato exercise a beneficial effect, such as the repression of othermicro-organisms present in the flora and interactions with the immunesystem of the intestine.

Relative Abundance of Microbiota

The methods include identifying at least one microbiota in a sample.Such a method for identifying a microbiota in a sample can includeproviding a sample comprising one or more microbiota, and detecting atleast one microbiota in the sample. One embodiment of the method mayinclude preparing a nucleic acid sample including a molecular indicatorof identity from at least one microbiota present in the sample anddetecting the molecular indicator of identity. For example, the methodcan involve preparing at least one nucleic acid sample by preparing aDNA sample. The molecular indicator of identity can be a polymorphicpolynucleotide, such as an rRNA gene (for example, a 16S rRNA gene). Themolecular indicator of identity can be detected by determining thenucleotide sequence of the polymorphic polynucleotide, such as the 16SrRNA gene, or a portion or subsequence thereof. Additional embodimentsfor detecting the molecular indicator of identity can also include PCRwith selective primers, quantitative PCR with selective primers, DNA-DNAhybridization, RNA-DNA hybridization, in situ hybridization, andcombinations thereof. For example, the polymorphic polynucleotide can bedetected by hybridization to a specific probe. In such an example, thespecific probe hybridizes to a polymorphic target nucleic acid, such asa 16S rRNA gene. Optionally, the nucleic acid can be hybridized to atleast one array comprising a plurality of specific probes, e.g., aplurality of specific probes, each of which identifies a microbiota.Detecting the molecular indicator of identity can also be accomplishedusing protein probes (such as antibodies) that bind to polymorphictarget proteins, for example polymorphic target proteins that identifythe microbiota.

The method of altering microbiota can also include measuring therelative abundance of one or more microbiota in a sample from a subject.As used herein, the term “relative abundance” refers to the commonalityor rarity of an organism relative to other organisms in a definedlocation or community. For example, the relative abundance can bedetermined by generally measuring the presence of a particular organismcompared to the total presence of organisms in a sample.

The relative abundance of microbiota can be measured directly orindirectly. Direct measurements can include culture based methods.Indirect measurements can include comparing the prevalence of amolecular indicator of identity, such as ribosomal RNA (rRNA) genesequences, specific for an organism or group of organisms in relation tothe overall sample. For example, a ratio of rRNA specific forBacteroidetes, Firmicutes, Proteobacteria, Tenericutes, orVerrucomicrobia in a total number of rRNA gene sequences obtained from acecal sample can be used to determine the relative abundance ofBacteroidetes, Firmicutes, Proteobacteria, Tenericutes, orVerrucomicrobia in the cecal sample.

In one embodiment, the relative abundance of microbiota, such asBacteroidetes, Firmicutes, Proteobacteria, Tenericutes, andVerrucomicrobia, within an individual subject may be calculated bymeasuring the ratio of one or more specific microbiota in a sample froman individual to obtain a microbiota profile of the subject. Therelative abundance can be derived from the total abundance of microbiotapresent in a sample. As used herein, the “total abundance” refersgenerally to the total organisms in a sample. As used herein,“microbiota profile” refers to a representation, such as a graph, of therelative abundance of one or more microbiota in a subject or sample froma subject.

The relative abundance of microbiota can be measured by obtaining asample from a subject. The sample can be saliva, feces, and stomach,intestinal and/or rectal content; tissue sample from a digestive tracttissue such as an oral tissue, esophagus, stomach, intestine, ileum,cecum, colon and/or rectum; an ascites within a gastrointestinal tissue;and any other sample that may be used by those familiar with assessingmicrobiota. In an exemplary embodiment, a relative abundance ofgastrointestinal microbiota is measured.

The methods can also include measuring a relative abundance ofmicrobiota in a biological sample, identifying one or more microbiota toproduce a microbiota profile of microbiota found in a particularsubject, e.g. individual having undergone a treatment or therapeuticintervention, or found in a particular location within the subject, e.g.gastrointestinal tract; and providing a composition comprising one ormore substantially purified microbiota selected from the microbiotaprofile, such as a Bacteroidetes, Firmicutes, Proteobacteria,Tenericutes, and Verrucomicrobia. In certain embodiments, the methodscan include identifying and categorizing microbiota in a subject.Typically, the identification is accomplished using culture-independentmethods. For example, as disclosed herein, the microbiota can beidentified by PCR using selective primers, quantitative PCR withselective primers, DNA-DNA hybridization, RNA-DNA hybridization and/orin situ hybridization. In some cases the hybridization is performed on amicroarray. Additionally, PCR or high-throughput sequencing methods candetect over- and under-represented genes in the total bacterialpopulation or transcriptomic or proteomic studies to identify lost orgained microbial transcripts or proteins within total bacterialpopulations. Alternatively, one or more species can be identified bydetermining the nucleotide sequence of a portion of a microbial genome,such as a 16S rRNA gene.

The methods can also include measuring total microbiota, individualmicrobiota taxa, such as phyla/classes/orders/families/genera/species,or measuring a combination of more than one microbiota taxa taken from atarget location, or at a specific time before and/or after an activity,such as ingesting food or physical activity, or pre- or post-treatment,such as a therapeutic intervention like pharmaceutical therapy, gastricbypass, duodenojejunal bypass, biliopancreatic diversion, verticalsleeve gastrectomy, adjustable gastric banding, vertical bandedgastroplasty, intragastric balloon therapy, gastric plication,Magenstrasse and Mill, small bowel transposition, biliary diversion,brown adipose tissue modulation (e.g., controlled activation, enhanceddifferentiation, supplemental implantation, etc.), pharmaceuticaladministration, electrical stimulation of nerves that innervate at leasta portion of the gastrointestinal tract, therapies impacting circadianrhythms, bile acid modulation, intestinal mucus production andmetabolism, and duodenal endoluminal barrier.

Individual relative abundances of microbiota may be obtained or totalabundances of microbiota may be obtained over an extended time period.The abundances of microbiota can include one or more of any of themicrobiota phyla/classes/orders/families/genera/species found in agastrointestinal tract of an animal (e.g., a human), and can beperformed by methods routinely used in the art including, by way of anon-limiting example, gastrointestinal tract content sampling. Therelative abundance or total abundances of microbiota may also includemeasuring total microbiota present in a sample.

The relative abundance of one or more microbiota levels can also bedetermined before, during or after an activity. The activities mayinclude, but are not limited to, physical activity, ingestion of food,and treatments, such as therapeutic interventions.

The relative abundance of one or more microbiota, a microbiota profile,can be compared to a target profile of relative abundances or totalabundance of microbiota. The target profile can be a standardizedmicrobiota profile obtained from one or more subjects of similar weight,age, gender, race, etc. The target profile can be a normalizedmicrobiota profile from a healthy subject of similar weight, age,gender, race, etc. The target profile can be a microbiota profile from ahealthy subject of similar weight, age, gender, race, etc that isresponsive or demonstrates a favorable outcome to a treatment ortherapeutic intervention. In a particular embodiment, the target profileis a microbiota profile from a healthy subject that is responsive to atherapeutic intervention, such as pharmaceutical therapy, gastricbypass, duodenojejunal bypass, biliopancreatic diversion, verticalsleeve gastrectomy, adjustable gastric banding, vertical bandedgastroplasty, intragastric balloon therapy, gastric plication,Magenstrasse and Mill, small bowel transposition, biliary diversion,brown adipose tissue modulation (e.g., controlled activation, enhanceddifferentiation, supplemental implantation, etc.), pharmaceuticaladministration, electrical stimulation of nerves that innervate at leasta portion of the gastrointestinal tract, therapies impacting circadianrhythms, bile acid modulation, intestinal mucus production andmetabolism, duodenal endoluminal barrier, or similar manipulations ofthe gastrointestinal tract.

The term “target profile” is intended to encompass any standard ornormal microbiota profile that can be useful as a benchmark againstwhich “altered microbiota profiles” can be measured. One skilled in theart can select a reference target profile in a myriad of ways so long asstatistically relevant measurements can be obtained. For example, atarget profile, or target profile for microbiota can be selected as theaverage level exhibited by healthy young adults (e.g., aged 25 to 30years old). Other standards or normal target profiles can be chosendepending upon the particular applications.

The relative abundance of one or more microbiota can be determinedthrough repeated microbiota profiles taken before, concurrent with,and/or after a metabolic disorder treatment, such as procedures likeadministration of a composition or agent like a weight loss supplement,pharmaceutical therapy, brown adipose tissue modulation (e.g.,controlled activation, enhanced differentiation, supplementalimplantation, etc.), pharmaceutical administration, electricalstimulation of nerves that innervate at least a portion of thegastrointestinal tract, therapies impacting circadian rhythms, bile acidmodulation, intestinal mucus production and metabolism, gastric bypass,duodenojejunal bypass, biliopancreatic diversion, vertical sleevegastrectomy, adjustable gastric banding, vertical banded gastroplasty,intragastric balloon therapy, gastric plication, Magenstrasse and Mill,small bowel transposition, biliary diversion, duodenal endoluminalbarrier, or similar manipulations of the gastrointestinal tract. Therepeated microbiota profiles can be used to compare relative abundanceof microbiota before, concurrent with, and/or after the treatment.Obtaining a microbiota profile after a treatment and comparing withpre-treatment profiles can also be used to determine or assessmicrobiota modifications that may be useful. The microbiota profile canbe obtained at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31days or more prior to the treatment. The microbiota profile can also beperformed at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 daysor more after the treatment. The microbiota profile can be obtainedconcurrently with the treatment.

Not only can the relative abundance of microbiota be obtained, butmeasurements of one or more other molecules that may contribute tovariations in relative abundance of microbiota, such as, but not limitedto, microbiota metabolites, microbiota components, glucoseconcentrations, leptin levels, or insulin levels, may be obtained. Themethod of measuring other molecules that may contribute to relativeabundance or total abundance of microbiota can include measuring themolecules in a sample from a subject. The sample can be the same sampleused to obtain the relative abundance of microbiota, or it can be adifferent sample. The sample can be, for example, a fecal, a blood, astomach content, and an intestinal content sample (e.g., luminal sample,mucus layer sample, mucosal adherent sample, etc.).

The methods and compositions may include altering the relative abundanceof one or more microbiota, such as Bacteroidetes, Proteobacteria,Firmicutes, Tenericutes, and Verrucomicrobia phyla or Bacteroidales,Enterobacteriales, Erysipelotrichales, Clostridiales, andVerrucomicrobiales orders or Alistipes, Escherichia, Clostridium,Allobaculum and Akkermansia genera, at least about a one-fold increaseor decrease. An exemplary embodiment may include a method forcomposition for altering the relative abundance of microbiota byadministering substantially purified microbiota, such as Bacteroidetes,Proteobacteria, Firmicutes, and Verrucomicrobia, to a subject. Forexample, substantially purified Bacteroidales, Enterobacteriales,Erysipelotrichales, Clostridiales, Verrucomicrobiales, Alistipes,Escherichia, Clostridium, Allobaculum, or Akkermansia may beadministered to a subject. The relative abundance of microbiota withinan individual subject may be altered (e.g., increased) from at leastabout 1% to at least about 1000% or more depending on the desired result(e.g., increased energy utilization (weight loss)) and the individualsubject. To treat a disorder, the relative abundance may be increased byat least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%,1000%, 2000%, 5000% or more. In an exemplary embodiment, the relativeabundance of Verrucomicrobia is increased by at least about 5%. Inanother embodiment, the relative abundance of at least one ofBacteroidetes, Proteobacteria, Firmicutes and Verrucomicrobia isincreased by at least about 5%. In another embodiment, the relativeabundance of at least one of Clostridia, Bacteroidales,Enterobacteriales, Clostridiales and Verrucomicrobiales is increased byat least about 5%. In another embodiment the relative abundance of atleast one of Erysipelotichales and Tenericutes is decreased by at leastabout 5%.

Another aspect encompasses a combination therapy to regulate fatstorage, energy utilization, and/or weight loss in a subject. In anexemplary embodiment, a combination for increasing energy utilization,decreasing body fat or for promoting weight loss may include combiningthe methods and compositions disclosed with a procedure or therapy suchas a pharmaceutical therapy, gastric bypass, duodenojejunal bypass,biliopancreatic diversion, vertical sleeve gastrectomy, adjustablegastric banding, vertical banded gastroplasty, intragastric balloontherapy, gastric plication, Magenstrasse and Mill, small boweltransposition, biliary diversion, brown adipose tissue modulation (e.g.,controlled activation, enhanced differentiation, supplementalimplantation, etc.), pharmaceutical administration, electricalstimulation of nerves that innervate at least a portion of thegastrointestinal tract, therapies impacting circadian rhythms, bile acidmodulation, intestinal mucus production and metabolism, duodenalendoluminal barrier or similar manipulations of the gastrointestinaltract. For example, a composition comprising a substantially purifiedmicrobiota, such as Bacteroidetes, Proteobacteria, Firmicutes,Verrucomicrobia, Bacteroidales, Enterobacteriales, Clostridiales,Verrucomicrobiales, Alistipes, Escherichia, Clostridium, or Akkermansiacan be administered to the subject prior to, concurrently with or aftera gastric bypass or other gastrointestinal or bariatric procedure.

Microbiota Compositions and Combination Compositions

Compositions are provided that can directly or indirectly alter therelative abundance of microbiota to a predetermined level, e.g. atherapeutic level, for a predetermined amount of time, e.g. until thenext dose is administered. The predetermined level can be obtained frommeasured relative abundances of microbiota that result in a therapeuticresponse, e.g. weight loss.

In one aspect, compositions can alter the relative abundance ofmicrobiota directly, such as through administration of microbiota in apharmaceutical composition or a food, drink, dietary supplement, and/orfood additive to be consumed by a subject. The compositions may includemicrobiota that are whole bacteria. Microbiota may also be viable(live), dormant, inactivated or dead bacteria. In an exemplaryembodiment, the composition includes a live bacterial strain ofmicrobiota from phyla such as Bacteroidetes, Proteobacteria, Firmicutesand Verrucomicrobia or orders such as Bacteroidales, Enterobacteriales,Clostridiales and Verrucomicrobiales or genera such as Alistipes,Escherichia, Clostridium, and Akkermansia.

The composition can also include mixtures of bacterial strains. Themixtures may include microbiota that are viable (live), dormant,inactivated or dead, or any combination thereof. In some embodiments,the microbiota can include a mixture or combination of live bacterialstrains.

A combination of bacteria from different phyla, such as Bacteroidetes,Firmicutes, Proteobacteria, Tenericutes, and Verrucomicrobia, can alsoexhibit a synergistic effect in certain applications (i.e. an effectwhich is greater than the additive effect of the bacteria when usedseparately). In addition, a combination of bacteria from differentphyla, different classes, different orders and different families can beuseful. For example, combinations which, in addition to having an effecton the mammal as single components, may influence other microbiota, e.g.by producing metabolites which are used as an energy source by othermicrobiota, or maintaining physiological conditions which favor ordisfavor other microbiota. In one embodiment, the composition includes amixture of at least two or more bacterial strains.

When the composition includes a combination of one or more microbiota,the microbiota can be from the same or differentphyla/classes/orders/families/genera/species. In an exemplaryembodiment, the composition includes at least one microbiota from phylasuch as Bacteroidetes, Proteobacteria, Firmicutes and Verrucomicrobia ororders such as Bacteroidales, Enterobacteriales, Clostridiales andVerrucomicrobiales or genera such as Alistipes, Escherichia,Clostridium, and Akkermansia. In another embodiment, the compositionincludes Verrucomicrobia, such as Verrucomicrobiales. In anotherembodiment, the composition includes Verrucomicrobia, such asAkkermansia. In yet another embodiment, the composition includesVerrucomicrobia and at least one or more of Bacteroidetes, Firmicutesand Proteobacteria. In another embodiment, the composition includesBacteroidetes, such as Bacteroidales. For example, the composition caninclude Alistipes. In another embodiment, the composition includesEnterobacteriales, such as Escherichia. In another embodiment, thecomposition can include Firmicutes, such as Clostridium.

In an exemplary embodiment, the composition can include bacteria fromthe class Verrucomicrobiae, bacteria from the order Verrucomicrobiales,bacteria from the family Verrucomicrobiaceae, and/or bacteria from thegenus Akkermansia. In another embodiment, the composition canadditionally include bacteria from at least one or more ofBacteroidetes, Firmicutes, and Proteobacteria. In another embodiment,the composition can include bacteria from the class Gammaproteobacteria,bacteria from the order Enterobacteriales, bacteria from the familyEnterobacteriaceae, and/or bacteria from the genus Escherichia. In yetanother embodiment, the composition can include bacteria from the classBacteroidia, bacteria from the order Bacteroidales, bacteria from thefamily Rikenellaceae, and/or bacteria from the genus Alistipes. Inanother embodiment, the composition can include bacteria from the classClostridia, bacteria from the order Lachnospiraceae, bacteria from thefamily Ruminococcaceae, and/or bacteria from the genus Clostridium.

In another exemplary embodiment, the composition can include bacteriafrom the class Bacteroidia, bacteria from the order Bacteroidales,bacteria from the family Rikenellaceae, and/or bacteria from the genusAlistipes. In another embodiment, the composition can additionallyinclude bacteria from at least one or more of Verrucomicrobia,Firmicutes, and Proteobacteria. In another embodiment, the compositioncan include bacteria from the class Gammaproteobacteria, bacteria fromthe order Enterobacteriales, and/or bacteria from the familyEnterobacteriaceae. In yet another embodiment, the composition caninclude bacteria from the class Verrucomicrobiae, bacteria from theorder Verrucomicrobiales, and/or bacteria from the familyVerrucomicrobiaceae. In another embodiment, the composition can includebacteria from the class Clostridia, bacteria from the orderLachnospiraceae, bacteria from the family Ruminococcaceae, and/orbacteria from the genus Clostridium.

In yet another embodiment, the composition can include bacteria from theclass Clostridia, bacteria from the order Clostridiales, bacteria fromthe family Lachnospiraceae, bacteria from the family Ruminococcaceae,and/or bacteria from the genus Clostridium. In another embodiment, thecomposition can additionally include bacteria from at least one or moreof Verrucomicrobia, Bacteroidetes, and Proteobacteria. In anotherembodiment, the composition can include bacteria from the classGammaproteobacteria, bacteria from the order Enterobacteriales, and/orbacteria from the family Enterobacteriaceae. In yet another embodiment,the composition can include bacteria from the class Verrucomicrobiae,bacteria from the order Verrucomicrobiales, and/or bacteria from thefamily Verrucomicrobiaceae. In yet another embodiment, the compositioncan include bacteria from the class Bacteroidia, bacteria from the orderBacteroidales, and/or bacteria from the family Rikenellaceae.

In another embodiment, the composition can include bacteria from two ormore phyla. For example, the composition can include bacteria from thephylum Bacteroidetes and bacteria from the phylum Verrucomicrobia. Inanother embodiment, the composition can include bacteria from two ormore classes. For example, the composition can include bacteria from theclass Clostridia and bacteria from the class Bacteroidia. In anotherexample, the composition can include bacteria from the class Clostridiaand bacteria from the class Verrucomicrobiae. In another example, thecomposition can include bacteria from two or more orders. For example,the composition can include bacteria from the order Bacteroidales andbacteria from the order Enterobacteriales. In another example, thecomposition can include bacteria from the order Verrucomicrobiales andbacteria from the order Enterobacteriales. In another embodiment, thecomposition can include bacteria from two or more genera. For example,the composition can include two or more of Alistipes, Escherichia,Clostridium, and Akkermansia.

The microbiota can also be substantially purified. The term“substantially purified” as used herein refers to a bacterial strain ora mixture of more than one bacterial strains (e.g., Bacteroidetes,Firmicutes, Proteobacteria, or Verrucomicrobia) that are substantiallyenriched in a sample. The sample can be substantially purified orenriched for the bacterial strain or mixture of strains of interest suchthat the sample is at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%or greater of the desired bacterial strain(s) or less than about 40%,30%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,1% or less of the undesirable or other bacterial strains present. In anexemplary embodiment, a composition includes substantially purifiedVerrucomicrobia. For example, the composition includes a substantiallypurified Akkermansia. In another embodiment, a composition includessubstantially purified Enterobacteriales. For example, the compositionincludes a substantially purified Escherichia. In another embodiment, acomposition includes substantially purified Bacteroidales. For example,the composition includes a substantially purified Alistipes. In anotherembodiment, a composition includes a substantially purified Clostridia.For example, the composition includes a substantially purifiedClostridium. In another exemplary embodiment, the composition includessubstantially purified Verrucomicrobia and at least one or more ofsubstantially purified Bacteroidetes, Firmicutes and Proteobacteria.Another embodiment is directed to a pharmaceutical composition foraltering microbiota including a therapeutically effective amount ofsubstantially purified microbiota, such as Verrucomicrobia,Bacteroidales, Clostridiales, Enterobacteriales, Alistipes, Escherichia,Clostridium, and/or Akkermansia.

Methods and compositions can also include treating weight relateddisorders, such as obesity, by substantially altering a relativeabundance of microbiota, by increasing or decreasing microbiota fromphyla such as Bacteroidetes, Proteobacteria, Firmicutes, Tenericutes andVerrucomicrobia or classes such as Mollicutes or orders such asBacteroidales, Enterobacteriales, Erysipelotrichales, Clostridiales andVerrucomicrobiales or genera such as Alistipes, Escherichia,Clostridium, Allobaculum and Akkermansia, in a gastrointestinal tract ofa subject without or in addition to a surgical procedure. In oneembodiment, the methods and compositions can include increasing arelative abundance of Verrucomicrobia and/or Enterobacteriales. Themethods and compositions can further include increasing a relativeabundance of Bacteroidetes. In another embodiment, the methods andcompositions can include increasing a relative abundance ofVerrucomicrobia, Bacteroidetes, Firmicutes, and/or Proteobacteria. In anexemplary embodiment, the methods and compositions can further includeincreasing a relative abundance of Bacteroidales, Enterobacteriales,Clostridia, Clostridiales, Verrucomicrobiae, Verrucomicrobiales,Alistipes, Escherichia, Clostridium, and/or Akkermansia. In anotherembodiment, methods and compositions can increase a relative abundanceof Enterobacteriales. In another embodiment, methods and compositionscan increase a relative abundance of Escherichia. In another embodiment,methods and compositions can increase a relative abundance of Alistipes.In another embodiment, methods and compositions can increase a relativeabundance of Clostridium. In another embodiment, methods andcompositions can increase a relative abundance of Akkermansia. In yetanother embodiment, the methods and compositions can include decreasinga relative abundance of Firmicutes and/or Tenericutes. In anotherembodiment, methods and compositions can decrease a relative abundanceof Erysipelotrichales. In another embodiment, methods and compositionscan decrease a relative abundance of Mollicutes. In another embodiment,methods and compositions can decrease a relative abundance ofAllobaculum.

Methods and compositions of altering a relative abundance of microbiotacan result in altered metabolic function. For example, alteringmetabolic function can include increasing energy expenditure and/orincreasing glucose metabolism. Energy expenditure can be increased by atleast about 5%, 6%, 7%, 8%, 9%, 10%, 15%, or 20%. The relative abundanceof microbiota can be altered such that at least one bacterial taxa hasan LDA of at least 2. In one embodiment, a relative abundance ofmicrobiota can be altered by increasing the relative abundance ofpropionate-producing bacteria in a gastrointestinal tract of a subject.For example, the relative abundance of E. coli can be increased.Propionate-producing bacteria can be increased such that the level ofpropionate in the gastrointestinal tract is increased by at least about50%, 60%, 70%, 80%, 90%, 99%, 100%, or %150%. In another embodiment, arelative abundance of microbiota can be altered to reduce acetate in agastrointestinal tract of a subject. In another embodiment, a ratio ofshort chain fatty acids can be altered in a gastrointestinal tract of asubject. For example, the ratio can be altered by manipulating bacteriain a gastrointestinal tract of a subject. Alternatively, the ratio canbe altered by creating bacteria to alter a ratio of short chain fattyacids in an environment. In another example, a cocktail of short chainfatty acids can be administered to a subject.

Another aspect includes compositions including compounds or agents thatalter the relative abundance of microbiota indirectly, such as throughthe administration of compound(s) or agent(s) that affect the growth,survival, persistence, transit or existence of one or more specificmicrobiota, such as Bacteroidetes, Firmicutes, Proteobacteria,Tenericutes and Verrucomicrobia. In one embodiment, the composition canincrease a relative abundance of bacteria. For example, the compositioncan increase a relative abundance of bacteria from the classBacteroidia, Gammaproteobacteria, Clostridia, and/or Verrucomicrobiae orbacteria from the order Bacteroidales, Enterobacteriales,Lachnospiraceae, and/or Verrucomicrobiales or bacteria from the familyRikenellaceae, Enterobacteriaceae, Clostridiaceae, and/orVerrucomicrobiaceae or bacteria from the genus Alistipes, Escherichia,Clostridium, and/or Akkermansia. In one embodiment, the composition candecrease a relative abundance of bacteria. For example, in oneembodiment, the composition can decrease the relative abundance ofbacteria from the class Erysipelotrichi, bacteria from the orderErysipelotrichales, and/or bacteria from the family Erysipelotrichaceae.In another example, the composition can decrease the relative abundanceof bacteria from the class Mollicutes and/or bacteria from the genusAllobaculum.

The compounds or agents can be antibiotic treatments and/orantibacterial agents. Antibiotics can also include naturally occurringantibacterial agents (e.g., magainins, defensins and others) orspecialized nutrient mixtures that alter the relative composition of themicrobiota. The compounds or agents can also be prebiotics. The term“prebiotic” refers to a component which increases the number ofprobiotic bacteria in the intestine. Thus, prebiotics as used herein mayrefer to any non-viable component that is specific to a bacteria thoughtto be of positive value, e.g. Verrucomicrobia. The administration of oneor more prebiotic compounds may selectively enhance the relativeabundance or general growth of one or more specific microbiota in vivoresulting in a pronounced health benefit, such as weight loss. Somenonlimiting examples of prebiotics can include bacterial cell wallcomponents such as peptidoglycans, bacterial nucleic acids such as DNAand RNA, bacterial membrane components, and bacterial structuralcomponents such as proteins, carbohydrates, lipids and combinations ofthese such as lipoproteins, glycolipids and glycoproteins. Additionalexamples can also include organic acids, inorganic acids, bases,proteins and peptides, enzymes and co-enzymes, amino acids and nucleicacids, carbohydrates, lipids, glycoproteins, lipoproteins, glycolipids,vitamins, bioactive compounds, metabolites containing an inorganiccomponent, small molecules, for example nitrous molecules or moleculescontaining a sulphurous acid, resistant starch, potato starch or highamylose starch, modified starches (including carboxylated starches,acetylated, propionated, and butyrated starches), non-digestibleoligosaccharides such as fructooligosaccharides, glucooligosaccharides,xylooligosaccharides, galactooligosaccharides, arabinoxylans,arabinogalactans, galactomannans, polydextrose, oligofructose, inulin,derivatives of these, but not excluding other oligosaccharides able toexert prebiotic effects, other soluble fibers, and combinations thereof.

The compounds or agents can also be provided in a food, drink, dietarysupplement, and/or food additive or can be used to modify a food, drink,dietary supplement, and/or food additive.

Yet another aspect can be directed to compositions that includecombinations of components that alter the relative abundance ofmicrobiota directly and indirectly. In one embodiment, a combinedadministration of microbiota with one or more compounds or agents, suchas prebiotics that foster the growth, survival, persistence, transit orexistence of one or more specific microbiota, otherwise termed as‘synbiotic’. The combination may result in a more pronounced healthbenefit, such as greater weight loss or faster weight loss.

The composition can be combinations of one or more microbiota, such asat least one microbiota from phyla such as Bacteroidetes,Proteobacteria, Firmicutes, and Verrucomicrobia or orders such asBacteroidales, Enterobacteriales, Clostridiales, and Verrucomicrobialesor genera such as Alistipes, Clostridium, Escherichia and Akkermansia,and one or more compounds or agents that foster the growth, survival,persistence, transit or existence of microbiota. The compounds or agentscan be antibiotic treatments and/or antibacterial agents, e.g., 25-50mg/kg/day for penicillin, 40-60 mg/kg/day for vancomycin, and 25-50mg/kg/day for tetracycline (see, e.g., Nelson's Pocket Book of PediatricAntimicrobial Therapy, 2002-2003, 15.sup.th Ed. J. Bradley & J. Nelson,eds., Lippincott Williams and Wilkins), prebiotics including bacterialcomponents such as bacterial cell wall components such as peptidoglycan,bacterial nucleic acids such as DNA and RNA, bacterial membranecomponents, and bacterial structural components such as proteins,carbohydrates, lipids and combinations of these such as lipoproteins,glycolipids and glycoproteins, bacterial metabolites, organic acids,inorganic acids, bases, proteins and peptides, enzymes and co-enzymes,amino acids and nucleic acids, carbohydrates, lipids, glycoproteins,lipoproteins, glycolipids, vitamins, bioactive compounds, metabolitescontaining an inorganic component, and small molecules such as nitrousmolecules or molecules containing a sulphurous acid, resistant starch,potato starch or high amylose starch, modified starches (includingcarboxylated starches, acetylated, propionated, and butyrated starches),non-digestible oligosaccharides such as fructooligosaccharides,glucooligosaccharides, xylooligosaccharides, galactooligosaccharides,arabinoxylans, arabinogalactans, galactomannans, polydextrose,oligofructose, inulin, derivatives of these, but not excluding otheroligosaccharides able to exert prebiotic effects, other soluble fibers,and combinations thereof.

The above compositions can also include, for example, amino acids, aminosugars, sugar alcohols, proteins, saccharides, di-saccharides,oligo-saccharides, poly-saccharides, nucleic acids, buffers,surfactants, lipids, liposomes, other excipients, and mixtures thereof.Other useful components can include steroids, anti-inflammatory agents,non-steroidal anti-inflammatory agents, analgesics, cells,anti-inflammatory agents, growth factors, growth factor fragments,small-molecule wound healing stimulants, hormones, cytokines, peptides,antibodies, enzymes, isolated cells, platelets, immunosuppressants,nucleic acids, cell types, viruses, virus particles, essentialnutrients, minerals, metals, or vitamins, and combinations thereof.Additionally, the composition can include a diluent, such as water,saline, or a buffer.

Screening for Compounds or Agents that Alter Microbiota

As disclosed above, a variety of compounds or agents can be useful foraltering microbiota in a subject. Such compounds or agents can includebut are not limited to antibiotic treatments and/or antibacterialagents, prebiotics such as bacterial cell wall components, bacterialnucleic acids such as DNA and RNA, bacterial membrane components, andbacterial structural components such as proteins, carbohydrates, lipidsand combinations of these such as lipoproteins, glycolipids andglycoproteins, organic acids, inorganic acids, bases, proteins andpeptides, enzymes and co-enzymes, amino acids and nucleic acids,carbohydrates, lipids, glycoproteins, lipoproteins, glycolipids,vitamins, bioactive compounds, metabolites containing an inorganiccomponent, small molecules, for example nitrous molecules or moleculescontaining a sulphurous acid, resistant starch, potato starch or highamylose starch, modified starches (including carboxylated starches,acetylated, propionated, and butyrated starches), non-digestibleoligosaccharides such as fructooligosaccharides, glucooligosaccharides,xylooligosaccharides, galactooligosaccharides, arabinoxylans,arabinogalactans, galactomannans, polydextrose, oligofructose, inulin,derivatives of these, but not excluding other oligosaccharides able toexert prebiotic effects, other soluble fibers, and combinations thereof.

The methods provided herein also can include methods of screening forand testing compounds' or agents' ability to alter the relativeabundance of select microbiota in a subject. Some of the methodsprovided herein are screening methods for testing the ability of apharmaceutical composition to alter microbiota from phyla such asBacteroidetes, Proteobacteria, Firmicutes, Tenericutes, andVerrucomicrobia or orders such as Bacteroidales, Enterobacteriales,Erysipelotrichales, Clostridiales and Verrucomicrobiales or genera suchas Alistipes, Escherichia, Clostridium, Allobaculum and Akkermansia.Other methods provided herein also can include testing compounds oragents in a food, drink, dietary supplement, and/or food additive forthe ability to alter microbiota. Such methods thereby identify apharmaceutical composition or a food, drink, dietary supplement, and/orfood additive that is capable of increasing or decreasing the relativeabundance of one or more microbiota from phyla such as Bacteroidetes,Proteobacteria, Firmicutes, Tenericutes, and Verrucomicrobia or orderssuch as Bacteroidales, Enterobacteriales, Erysipelotrichales,Clostridiales and Verrucomicrobiales or genera such as Alistipes,Escherichia, Clostridium, Allobaculum and Akkermansia.

Any of a variety of diagnostic factors can be monitored as indicators ofefficacy, such as those known in the art. For example, weight changes,blood pressure, serum insulin/glucose levels, energy expenditure,breathing, color, temperature or other diagnostic indicators that can bemeasured to determine efficacy of the compound or agent. In addition,the presence or absence or level of one or more components in a samplefrom a subject can also be factors for determining efficacy of thecompound or agent. Typical samples can include blood and urine samples,where the presence or absence or level of one or more components can bedetermined by performing, for example, a blood panel or a urine paneldiagnostic test. Exemplary components indicative of a subject's healthinclude, but are not limited to, white blood cell count, hematocrit, andprotein concentration. In an exemplary embodiment, monitoring weightloss or alleviation of a disorder is used as an indicator of efficacy ofthe compound or agent as a pharmaceutical or food, drink, dietarysupplement, and/or food additive.

The methods can also include monitoring the relative abundance ofmicrobiota in a biological sample. For example, as disclosed herein, themicrobiota can be monitored by PCR using selective primers, quantitativePCR with selective primers, DNA-DNA hybridization, RNA-DNA hybridizationand/or in situ hybridization. Additionally, PCR or high-throughputsequencing methods can detect over- and under-represented genes in thetotal bacterial population or transcriptomic or proteomic studies toidentify lost or gained microbial transcripts or proteins within totalbacterial populations to monitor changes in relative abundance ofspecific microbiota from phyla such as Bacteroidetes, Proteobacteria,Firmicutes, Tenericutes, and Verrucomicrobia or orders such asBacteroidales, Enterobacteriales, Erysipelotrichales, Clostridiales andVerrucomicrobiales or genera such as Alistipes, Escherichia,Clostridium, Allobaculum and Akkermansia. Alternatively, the relativeabundance of one or more microbiota can be monitored by measuring therelative abundance of specific microbial genomes, such as a 16S rRNAgene, within total bacterial populations.

Provided herein is a kit for monitoring efficacy of an administeredtherapy as measured by changes in select microbiota. The kit can test asample from a subject for the relative abundance of one or moremicrobiota from phyla such as Bacteroidetes, Proteobacteria, Firmicutes,Tenericutes, and Verrucomicrobia or orders such as Bacteroidales,Enterobacteriales, Erysipelotrichales, Clostridiales andVerrucomicrobiales or genera such as Alistipes, Escherichia,Clostridium, Allobaculum and Akkermansia. In some embodiments, the kitcan contain reagents, devices, or components for detecting the relativeabundance of microbiota from phyla such as Bacteroidetes,Proteobacteria, Firmicutes, Tenericutes, and Verrucomicrobia or orderssuch as Bacteroidales, Enterobacteriales, Erysipelotrichales,Clostridiales and Verrucomicrobiales or genera such as Alistipes,Escherichia, Clostridium, Allobaculum and Akkermansia and a control.Exemplary kits can include reagents, devices, or components to measureindicators of efficacy as provided herein and can, optionally, includeone or more components such as instructions for use, devices, orcomponents for detecting the selected microbiota or the relativeabundance of the selected microbiota in a sample and, optionally, adevice for obtaining and/or processing a sample from a subject. In oneembodiment, the kit can include primers that selectively hybridize tonucleic acids from Verrucomicrobiales, Bacteroidales, Clostridiales,Erysipelotrichales, or Enterobacteriales species and hybridizationreagents.

Formulations

The disclosed compositions can be formulated as a pharmaceuticalcomposition. Such pharmaceutical compositions can include apharmaceutically acceptable carrier. An exemplary embodiment is directedto a pharmaceutical composition for altering microbiota including atherapeutically effective amount of substantially microbiota from phylasuch as Bacteroidetes, Proteobacteria, Firmicutes, Tenericutes, andVerrucomicrobia or orders such as Bacteroidales, Enterobacteriales,Erysipelotrichales, Clostridiales and Verrucomicrobiales or genera suchas Alistipes, Escherichia, Clostridium, Allobaculum and/or Akkermansia,and pharmaceutically acceptable carrier. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible. In one embodiment, formulations andcompositions of the present invention can be incorporated intopharmaceutical compositions suitable for delivery to a subject. Apharmaceutical composition may also comprise a pharmaceuticallyacceptable carrier. As used herein, “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like that are physiologically compatible. Examples ofpharmaceutically acceptable carriers include one or more of water,saline, phosphate buffered saline, dextrose, glycerol, ethanol and thelike, as well as combinations thereof. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, or sodium chloride in the composition.

The compositions can be formulated in a variety of forms. These include,for example, liquid, semi-solid and solid dosage forms, such as liquidsolutions (e.g., injectable and infusible solutions), dispersions orsuspensions, tablets, pills, powders, liposomes, suppositories, andother formulations. The compositions can also be formulated for highdrug concentrations. The compositions can further be sterile and stableunder the conditions of manufacture and storage. Sterile injectablesolutions can be prepared by incorporating the compositions in arequired amount of an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization.

Exemplary forms of the compositions can depend on the intended mode ofdelivery and therapeutic application. In one embodiment, the compositionis formulated for oral delivery. Some compositions can be in the form ofpill-based delivery, such as disclosed in U.S. patent application Ser.No. 12/976,648 entitled “Pill Catcher,” filed Dec. 22, 2010, and delayedrelease methods. In one embodiment, the pill-based delivery can be partof the system that allows the delivery to occur at a precise locationwithin the gastrointestinal tract. In another embodiment, thecompositions can be formulated in a delayed release formulation. Inanother embodiment, the composition can be encapsulated in a coatingthat does not begin to degrade until it exits the exits the stomach of apatient. In another embodiment, the composition can be prepared with acarrier that will protect the composition against rapid release, such asa controlled release formulation, including implants, transdermalpatches, and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Many methods for the preparation of such formulationsare patented or generally known to those skilled in the art. See, e.g.,Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson,ed., Marcel Dekker, Inc., New York, 1978. “Sustained release” refers torelease of a composition or an active compound thereof over a prolongedperiod of time relative to that achieved by delivery of a conventionalformulation of the composition.

Another type of composition includes activatable forms, such asformulating a composition with microbiota in a dormant or inactivestate, such as, a lyophilized state. In combination compositions, themicrobiota may be in a dormant or inactive state or the compounds oragents that foster microbiota can be inactive. In an exemplaryembodiment, the composition is formulated to include at least one of adormant or inactive microbiota and inactive compounds or agents thatfoster microbiota.

The disclosed compositions and combination compositions can also beformulated as a food, drink, dietary supplement, and/or additive. Suchcompositions are those that are suitable for human and/or animalconsumption. A skilled person will be readily aware of specificformulations of the microbiota which can be used in oral or ingestibleformulations and are considered suitable for human and/or animaladministration. Many compositions are used for the manufacture of foodor food additive/supplemental; consequently a further important aspectis the provision of a human or animal food or food additive/supplementalincluding microbiota from phyla such as Bacteroidetes, Proteobacteria,Firmicutes and Verrucomicrobia or orders such as Bacteroidales,Enterobacteriales, Clostridiales, and Verrucomicrobiales or genera suchas Alistipes, Escherichia, Clostridium, and Akkermansia, to increaseweight loss in a mammal.

Consumable compositions can be formulated to include a sweetener(s), astabilizer(s) or binder(s), a humectant(s), and/or natural and/orartificial flavors. The compositions may also include natural and/orartificial colors and preservatives. In one implementation, thecompositions may include mono-saccharides, di-saccharides andpoly-saccharides such as but not limited to, sucrose (sugar), dextrose,maltose, dextrin, xylose, ribose, glucose, mannose, galactose,sucromalt, fructose (levulose), invert sugar, corn syrups,maltodextrins, fructo oligo saccharide syrups, partially hydrolyzedstarch, corn syrup solids, polydextrose, soluble fibers, insolublefibers, natural cane juice, gelatin, citric acid, lactic acid, naturalcolors, natural flavors, fractionated coconut oil, carnauba wax, orcombinations thereof.

Dosage

The microbiota compositions can also include a “therapeuticallyeffective amount,” or an “effective amount.” A “therapeuticallyeffective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic result. Atherapeutically effective amount of a composition may vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of the composition to elicit a desiredresponse in the individual. A therapeutically effective amount is alsoone in which any toxic or detrimental effects of the composition areoutweighed by the therapeutically beneficial effects. In an exemplaryembodiment, a therapeutically effective amount of the microbiotacomposition is one in which the amount increases a relative abundance ofone or more microbiota. For example, the therapeutically effectiveamount of Verrucomicrobia increases a relative abundance ofVerrucomicrobia in a subject.

The dosage of the compositions can be dependent on the types ofmicrobiota present in the composition. The dosage can also be determinedbased on a relative abundance of one or more microbiota present in thesubject. The dosage can also be determined by additional treatments ortherapeutic interventions, such as procedures like administration of acomposition or agent like a weight loss supplement, pharmaceuticaltherapy, brown adipose tissue modulation (e.g., controlled activation,enhanced differentiation, supplemental implantation, etc.),pharmaceutical administration, electrical stimulation of nerves thatinnervate at least a portion of the gastrointestinal tract, therapiesimpacting circadian rhythms, bile acid modulation, intestinal mucusproduction and metabolism, gastric bypass, duodenojejunal bypass,biliopancreatic diversion, vertical sleeve gastrectomy, adjustablegastric banding, vertical banded gastroplasty, intragastric balloontherapy, gastric plication, Magenstrasse and Mill, small boweltransposition, biliary diversion, duodenal endoluminal barrier, orsimilar manipulations of the gastrointestinal tract.

In one embodiment, the composition can be effective to alter therelative abundance of one or more microbiota. In another embodiment, thecomposition can be effective to increase a relative abundance of themicrobiota in a subject. In one embodiment, the composition can increaseor decrease a relative abundance of a specific strain of microbiota fromphyla such as Bacteroidetes, Proteobacteria, Firmicutes, Tenericutes,and Verrucomicrobia or orders such as Bacteroidales, Enterobacteriales,Erysipelotrichales, Clostridiales and Verrucomicrobiales or genera suchas Alistipes, Escherichia, Clostridium, Allobaculum, and Akkermansia, ina subject. In an exemplary embodiment, the composition can increase arelative abundance of microbiota from phyla such as Bacteroidetes,Proteobacteria, Firmicutes, and Verrucomicrobia or orders such asBacteroidales, Verrucomicrobiales, Clostridiales and Enterobacterialesor genera such as Alistipes, Escherichia, Clostridium, and Akkermansiain a subject.

In another embodiment, the composition can also be effective to altermicrobiota in the subject so that after administration of thecomposition the microbiota in the subject mimics a microbiota found in asubject responsive to a gastric bypass or other gastrointestinal orbariatric procedure. The composition can be effective to altermicrobiota to mimic microbiota from normal, healthy subjects of similarweight, age, gender, race, etc. In an exemplary embodiment, themicrobiota can be altered, increased or decreased, to mimic one or moresubjects of similar weight, age, gender, race, etc that are responsiveor demonstrate a favorable outcome to a surgical procedure ortherapeutic intervention, such as gastric bypass or othergastrointestinal bariatric or metabolic procedure, for the treatment ofobesity, diabetes, other metabolic disorders or comorbidities ofobesity. In an exemplary embodiment, the composition can be effective toalter microbiota in the subject to mimic microbiota found in a subject,such as a subject that is responsive to a surgical procedure likegastric bypass or other gastrointestinal bariatric or metabolicprocedures.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be delivered, several divided doses may be delivered over timeor the dose may be proportionally reduced or increased as indicated bythe exigencies of the therapeutic situation. It is especiallyadvantageous to formulate parenteral compositions in dosage unit formfor ease of delivery and uniformity of dosage. Dosage unit form as usedherein refers to physically discrete units suited as unitary dosages forthe mammalian subjects to be treated; each unit containing apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeutic orprophylactic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofindividuals.

An exemplary dosage of a composition when employed in the methodprovided can be in the range from about 0.001 to about 100 mg/kg bodyweight per day, from about 0.01 to about 50 mg/kg body weight per day,such as from about 0.05 to about 10 mg/kg body weight per day, deliveredin one or most doses, such as from 1 to 3 doses. In an exemplaryembodiment, the composition includes substantially purifiedVerrucomicrobia or one or more of substantially purifiedVerrucomicrobia, Bacteroidetes, Firmicutes and Proteobacteria in therange of about 0.01 to about 50 mg/kg body weight per day, delivered inone to three doses. The exact dosage will depend upon the frequency andmode of delivery, the gender, age, weight and general condition of thesubject treated, the nature and severity of the condition treated, anyconcomitant diseases to be treated and other factors evident to thoseskilled in the art.

In one embodiment, composition includes substantially purifiedmicrobiota, such as Verrucomicrobia, Bacteroidetes, Firmicutes and/orProteobacteria, at a total concentration in the range of about 0.001mg/kg to about 100 mg/kg. In another embodiment, the compositionincludes substantially purified microbiota, such as Verrucomicrobia,Bacteroidetes, Firmicutes and/or Proteobacteria, at a totalconcentration in the range of about 0.1 mg/kg to about 50 mg/kg. In yetanother embodiment, the composition includes substantially purifiedmicrobiota, such as Verrucomicrobia, Bacteroidetes, Firmicutes and/orProteobacteria, at a total concentration in the range of about 1 mg/kgto about 10 mg/kg.

Delivery

The microbiota composition can be delivered or administered by a varietyof methods known in the art. The terms “delivery,” “deliver,”“administration” and “administer” are used interchangeable herein. Aswill be appreciated by the skilled artisan, the route and/or mode ofdelivery will vary depending upon the desired results. In oneembodiment, the microbiota composition is delivered perorally. Inanother embodiment, the microbiota composition is delivered orally. Yetanother mode of delivery can include methods and combinations fordelivery to the gut.

The microbiota composition can be delivered to target regions and/orstructures within the subject. Regions that can be targeted within thegastrointestinal tract can include, but are not limited to, the stomach,biliopancreatic limb, Roux limb, common limb, ileum, cecum, or colon.Structures can be targeted that constitute differentiated ecologicalniches with specific pH range, temperature, moisture, and metabolitecontent. Diseases and conditions associated with altered microbialprofiles may exhibit either the presence of a novel microbe(s), absenceof a normal microbe(s), or an alteration in the proportion of microbes.

Delivery of the microbiota composition can be targeted to one or moreregions in a subject. The regions can include but are not limited to aregion within the gastrointestinal tract. In an exemplary embodiment,the delivery is targeted to an oral cavity, stomach, biliopancreaticlimb, Roux limb, common limb, small intestine, ileum, cecum, largeintestine, or colon of a gastrointestinal tract. The delivery can alsobe targeted to one or more tissues in a subject. The tissues can includeany tissue in a gastrointestinal tract, such as a stomach,biliopancreatic limb, Roux limb, common limb, small intestine, ileum,cecum, large intestine, or colon.

The composition can be delivered before, current with or after atherapeutic treatment, such as procedures like administration of acomposition or agent like a weight loss supplement, pharmaceuticaltherapy, brown adipose tissue modulation (e.g., controlled activation,enhanced differentiation, supplemental implantation, etc.),pharmaceutical administration, electrical stimulation of nerves thatinnervate at least a portion of the gastrointestinal tract, therapiesimpacting circadian rhythms, bile acid modulation, intestinal mucusproduction and metabolism, gastric bypass, duodenojejunal bypass,biliopancreatic diversion, vertical sleeve gastrectomy, adjustablegastric banding, vertical banded gastroplasty, intragastric balloontherapy, gastric plication, Magenstrasse and Mill, small boweltransposition, biliary diversion, duodenal endoluminal barrier, orsimilar manipulations of the gastrointestinal tract. In one embodiment,the composition can be delivered at least about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31 days or more before the treatment. In anotherembodiment, the composition can be delivered at least about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31 days or more after the treatment. In yetanother embodiment, the composition can be delivered concurrently withthe therapeutic treatment. In another embodiment, the composition can bedelivered for a treatment period that lasts until the desired outcome(e.g., weight loss, diabetes improvement, target population has beenachieved, the target population has been maintained, etc.) is achieved.

Delivery of the microbiota composition can also be repeated one or moretimes. The repeated delivery of the microbiota composition can be one ormore times before and/or after a metabolic disorder treatment. Therepeated delivery can be in a manner similar to the initial delivery.

The microbiota composition can also be administered with agents that mayinclude therapeutic, prophylactic, or diagnostic agents selected fromsmall molecules, nucleic acids, proteins, prebiotics like polypeptides,prebiotics including bacterial components such as bacterial cell wallcomponents such as peptidoglycan, bacterial nucleic acids such as DNAand RNA, bacterial membrane components, and bacterial structuralcomponents such as proteins, carbohydrates, lipids and combinations ofthese such as lipoproteins, glycolipids and glycoproteins, bacterialmetabolites, organic acids, inorganic acids, bases, proteins andpeptides, enzymes and co-enzymes, amino acids and nucleic acids,carbohydrates, lipids, glycoproteins, lipoproteins, glycolipids,vitamins, bioactive compounds, metabolites containing an inorganiccomponent, and small molecules such as nitrous molecules or moleculescontaining a sulphurous acid, resistant starch, potato starch or highamylose starch, modified starches (including carboxylated starches,acetylated, propionated, and butyrated starches), non-digestibleoligosaccharides such as fructooligosaccharides, glucooligosaccharides,xylooligosaccharides, galactooligosaccharides, arabinoxylans,arabinogalactans, galactomannans, polydextrose, oligofructose, inulin,derivatives of these, but not excluding other oligosaccharides able toexert prebiotic effects, other soluble fibers, and combinations thereof.In one embodiment, the agent delivered is a small molecule deliveredthat has low oral bioavailability and acts on a microbial niche of thehost's gut. Low oral bioavailability is generally undesirable in drugs,since absorption through the intestine is an objective of most oraltherapies.

The microbiota composition can also be administered in the samecomposition with compounds or agents as described above or can beadministered individually with the compounds or agents administeredbefore, concurrent with, and/or after the microbiota compositions. Themicrobiota composition can be administered at least about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31 days or more prior to the administration ofcompounds or agents. The microbiota composition can also be administeredat least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 days or moreafter the administration of compounds or agents. The microbiotacomposition can be administered concurrently with the administration ofcompounds or agents.

The composition can also be delivered by a system that can be at leastpartially implantable. The implantable system can be any of those knownand used in the art. The system can include a programmable pump such asthose commonly used to deliver insulin to a diabetic patient. One ormore of these components may be modular and connected to atranscutaneous delivery system which may include a port, needle, patch,or the like. In an exemplary embodiment, the implantable system includesa reservoir and a port. The reservoir may include a refillable orreloadable container for holding the composition. In another embodiment,the system can include a catheter. In another embodiment, theimplantable system is a translumenal catheter. The system can also beconfigured to deliver the composition at a prescribed dosage and/or aprescribed interval. The prescribed dosage and/or prescribed intervalcan be determined by those of skilled in the art.

One skilled in the art will appreciate further features and advantagesof the invention based on the above-described embodiments. Accordingly,the invention is not to be limited by what has been particularly shownand described in the examples or figures, except as indicated by theappended claims. All publications and references cited herein areexpressly incorporated herein by reference in their entirety.

EXAMPLES Example 1 Materials and Methods

Animals.

Male C57BL/6J diet-induced obese (DIO) mice were purchased at 22-26weeks of age from Jackson Laboratories. These mice were maintained on a60% high fat diet (HFD; Research Diets, D12492, New Brunswick, N.J.)from 6 weeks of age until they reached a surgical weight of 40-50 grams.All animal studies were performed in accordance with the MassachusettsGeneral Hospital Subcommittee on Research Animal Care guidelines andused under an approved protocol. Male, age-matched (7-10 weeks old),germ-free Swiss Webster mice were obtained from Taconic and used underan approved Harvard Medical School IACUC protocol.

Animals in the RYGB group were fasted overnight and anesthetized withisofluorane. The abdomen was exposed and a single transection was madejust distal to the ligament of Treitz. The proximal intestinal portion(the biliopancreatic (BP) limb) was re-anastamosed to a jejunal segment˜9 cm (6 Q-tips) distal to the transection to create the “Y” junction.The glandular and non-glandular portion of the stomach wasdouble-sutured and transected to form the stomach remnant and stomachpouch, respectively. An incision was made at the stomach pouch and thedistal segment of the transection intestine (proximal Roux (Rx) limb)was anastamosed to the stomach. All anastamoses were double-checked forleakage prior to body wall and subcutaneous closures.

Sham-operated animals (SHAM) were treated in a similar manner as theRYGB animals pre-operatively, and the same intestinaltransection-anastomosis was performed. The stomach was manipulated in asimilar manner as the RYGB animals, but no transections or incisionswere made of the stomach. See FIG. 2.

All animals were given 0.05 mg/kg buprenorphine IM for prevention and/orrelief of post-operative pain. After surgery, animals were housedindividually on wire floors and maintained on liquid diet (Vital H N,Abbott Labs, Columbus, Ohio) for up to 2 weeks until fully weaned backonto high fat diet (HFD). Body weights were monitored weekly. Threeweeks post-operatively, a group of weight-stabilized SHAM animals wererandomly chosen and maintained on a daily restricted diet of ˜⅓ of theRYGB daily food intake in order to reduce and maintain body weight to anormal weight of ˜30-33 grams to match the weight of the RYGB animals(weight-matched sham group; WMS). See FIG. 2. For indirect calorimetryexperiments, animals were fed a powdered, irradiated form of Mouse Diet9F (Labdiet 5020).

Fecal Sampling and Processing.

Weekly fecal samples were collected directly into DNase- and RNase-freetubes and stored at −80° C. until processed. DNA was extracted using thePowerSoil bacterial DNA extraction kit (MoBio, Carlsbad Calif.) andPCR-amplified using universal bacterial primers targeting variableregion 4 of the 16S rRNA gene with the following thermocylcer protocol:Denature at 94° C. for 3 min, 35 cycles of 94° C. for 45 sec, 50° C. for30 sec, and 72° C. for 90 sec, with final annealing temperature at 72°C. for 10 min. Triplicate reactions for each sample were pooled andamplification was confirmed with gel electrophoresis on a 1.5% agarosegel. 16S rRNA DNA amplicons were cleaned with the Ampure XP kit(Agencourt, Danvers, Mass.) and quantified using the Quant-iT Picogreends DNA Assay Kit (Invitrogen, Carlsbad, Calif.). Amplicons weresequenced with the Illumina Hi-Seq platform. Multivariable statisticalanalysis was used to compare microbial composition among the treatmentgroups. 16S rRNA gene sequences were analyzed using the QIIME softwarepackage (Quantitative Insights Into Microbial Ecology) in addition tocustom Perl scripts to analyze alpha (within sample) and beta (betweensample) diversity.

Food Intake and Fecal Analysis.

A subgroup of animals was used for food intake and fecal analysis.Animals were housed individually on wire floors. Food was weighed everyother day and adjusted for spillage. All fecal pellets were collected,weighed, and submitted to the University of Arkansas for fecalcalorimetry, crude fat, and crude nitrogen. Sample amount permitting,fecal pH was also measured.

Tissue Harvest and Intestinal Axis Sampling.

Animals were sacrificed between 12 and 15 weeks after surgery. Prior toeuthanization, animals were fasted for 2 hours, and a blood glucoselevel was measured from the tail tip using a handheld glucometer(Alpha-Trak, Abbott Labs; Abbott Park, Ill.), anesthetized with 100mg/kg IP sodium pentobarbital and euthanized by cardiac exsanguination.Blood was collected in EDTA-containing tubes and plasma was stored at−80° C. The following intestinal sections were collected for bacterialDNA analysis: stomach pouch, stomach remnant, BP limb, Roux limb, commonlimb (first 3 cm distal to BP/Rx anastomosis), the distal ileum, ½cecum, and colon. Sections representative of each limb were alsocollected in SHAM and WMS animals. Each section was flushed withextraction buffer (200 mM NaCl, 200 mM Tris, 20 mM EDTA, pH 8.0) toretrieve luminal bacterial contents. Each section was then cutlongitudinally and the mucosa scraped off with ethanol-cleanedmicroscope slides to obtain mucosal adherent bacteria samples.Collection of ceca and colons were performed separately from the rest ofthe intestine to avoid contamination. All contents and mucosal adherentsamples were flash frozen in liquid nitrogen and stored at −80° C. untilDNA extraction with the same PowerSoil DNA extraction kit mentionedabove. In another subgroup of animals, pH was measured in differentsegments of the gastrointestinal tract, using a pH meter connected to amicro pH electrode (Thermo Scientific, Rochester, N.Y.). In addition tointestinal sampling, epididymal and retroperitoneal fat pads werecollected and weighed as a biomarker for visceral adiposity. Whole bodylean and fat mass were determined by NMR (Bruker T D Minispec,Billerica, Mass.).

Biochemical Assays.

Plasma insulin was measured using the mouse ultrasensitive insulin ELISA(Alpco, Salem, N.H.). Fasting TG and non-esterified fatty acids (NEFAs)were assayed from serum using their respective kits (Wako Chemical,Richmond, Va.). Liver triglycerides were extracted with overnightincubation in ethanolic KOH at 55° C., and free glycerol was measuredusing free glycerol reagent (Sigma, Saint Louis, Mo.). Plasma leptin wasmeasured by ELISA (Crystal Chem, Inc., Downers Grove, Ill.).

Germ-Free Mouse Experiments.

A donor animal from each group—RYGB, sham operated (SHAM), andnon-operated diet induced obese (DIO) mice—was treated in the same wayas all other mice, except that the cecal contents were saved and mixedin reduced anaerobic PBS buffer. The sample was homogenized in ananaerobic chamber, and the resultant slurry administered by oral gavageto germ-free, 7-13 week old, male Swiss Webster recipient mice (5-6animals/group). Animals were housed 1-2/cage on wire floors and fedautoclaved rodent breeder chow. A group of uninoculated mice were usedas a control. Body weights and cumulative food intake were recordedweekly during the 2-week colonization period. Fecal pellets werecollected at days −1, 1, 2, 3, 7, and 13 after gavage. On day 13, foodwas withheld from animals overnight. On day 14, animals were removedfrom the microisolators and transferred to a procedure room, where afasting blood glucose was taken with a handheld glucometer (Alpha-trak,Abbott Labs) from blood taken from the tail tip. Animals wereanaesthetized with isofluorane. A terminal blood collection was taken,tissues harvested, and liver and visceral (retroperitoneal andepididymal) fat pads were weighed.

Doubly Labeled Water.

Doubly=labeled water was made using a 3:1 dilution of 0-18 water (97%;Cambridge Isotope Laboratories, Andover, Mass.; OLM-240-97-1) anddeuterium oxide (Sigma, #151882-10G), sterile filtered, and stored inethylene oxide sterilized amber injection bottles with crimped lids. Apre-injection baseline sample was collected 1-2 days prior to theexperiment. Following oral gavage of donor sample microbiota, animalswere injected with 60 μL (66 μg) of doubly-labeled water tracer IP andthe time of injection was recorded. A post-injection blood sample wascollected between 60-90 minutes following injection and used as themaximum equilibrated tracer dilution. Blood samples were then collectedat days 5, 8, and 14 following injection. Times were precisely recordedwith each blood sample collection. Blood samples were centrifuged at10,000 rcf for 5 minutes to separate plasma and stored at −80 C untilanalysis. Samples from days −1, 0, 5, and 8 were sent to MetabolicSolutions (Andover, Mass.) for tracer analysis.

To determine the rate of CO₂ production (rCO₂), the log of the deuteriumand O-18 levels of blood samples from each animal were plotted againsttime to retrieve a linear slope, representing the rate of tracerelimination from the body pool. These samples were compared with aninternational standard (Vienna Standard Mean Ocean Water). The rate ofCO2 production (rCO2) was calculated based on modified equations ofSpeakman, Nair, and Goran (Am J Physiol 264:E912-E917, 1993):

rCO₂=(N/2.196)×(k _(o)−1.0472k _(d)) where N=[(N_(o))+(N_(d)/1.0427)]/2

The estimate of energy expenditure was calculated with an RQ estimate of0.85. Total body water was determined based on the mean pool sizes fordeuterium and oxygen-18.

Indirect Calorimetry.

Animals were transported in sterile containers prior to placement in a16 cage CLAMS (Comprehensive Lab Animal Monitoring System; Columbus,Ohio) and left to sit for 1 hour prior to start of measurements. Flowrate was set to 0.6 L/min. Animals were checked daily for food andwater; two animals required refilling of food. One GF-R animal chewedmuch of the food into the spillage bucket and required refills duringboth days; an error in re-balancing the food weigh scale produced anartificial increase in food intake. Thus, the animal's food intake datawas taken out of the study.

Short Chain Fatty Acid Analysis.

Cecal content samples were kept frozen at −80° C. until analysis. Thesamples were removed from the freezer and weighed, and 500 μL of water(HPLC grade) was added to each of the thawed samples. The samples werevortexed for 1 minute until the material was homogenized. The pH of thesuspension was adjusted to 2-3 by adding 50 μL of 50% sulfuric acid. Theacidified samples were kept at room temperature for 5 minutes andvortexed briefly every minute. The samples were then centrifuged for 10minutes at 5000 g. 400 μL of the clear supernatant was transferred intoan Eppendorf tube for further processing. For the volatile extraction,50 μL of the internal standard (1% 2-methyl pentanoic acid solution) and400 μL of ethyl ether anhydrous were added. The tubes were vortexed for30 seconds and then centrifuged at 5000 g for 10 minutes. 1 μL of theupper ether layer was injected into the chromatogram for analysis andcompared with an internal standard control solution containing 10 mM ofacetic, propionic, isobutyric, butyric, isovaleric, valeric, isocaproic,caproic, and heptanoic acids (Matreya, Pleasant Gap Pa.).

Chromatographic analysis was carried out using a Shimadzu GC14-A systemwith a flame ionization detector (FID) (Shimadzu Corp, Kyoto, Japan). Afused silica capillary column 30 m×0.25 mm coated with 0.25 um filmthickness was used (Nukol™) for the volatile acids (Supelco Analytical,Bellefonte, Pa.). Nitrogen was used as the carrier gas. The oventemperature was 170° C. and the FID and injection port was set to 225°C. The injected sample volume was 1 μL and the run time for eachanalysis was 10 minutes. The chromatograms and data integration wascarried out using a Shimadzu C-R5A Chromatopac. The retention times andpeak heights of the acids in the standard mix were used as referencesfor the sample unknowns. These acids were identified by their specificretention times and the concentrations determined and expressed as mMconcentrations per gram of sample.

RNA Extraction and qPCR.

RNA was extracted from most tissues with Trizol (Invitrogen, Carlsbad,Calif.). Adipose tissue RNA was extracted using the lipid tissue RNeasyQiagen kit (Valencia, Calif.). 1 μg of RNA was used for cDNAamplification, using the SuperScript III First-Strand Synthesis Systemand random hexamer primers under manufacturer instructions (Invitrogen,Carlsbad, Calif.). qPCR was performed with 50 ng of cDNA usinginventoried primers from Applied Biosystems (Carlsbad, Calif.). Geneexpression was analyzed by the ΔΔC_(T) method (Applied BiosystemsBulletin #2) with the appropriate control condition as calibrator.Beta-actin was used as a housekeeping gene.

Statistical Analysis.

For in vivo physiology experiments, all data are expressed as means±SEM.Statistics were performed using GraphPad Prism (v. 5). Significance wasaccepted at P<0.05.

Example 2 Donor Analysis

Mice were separated into three groups: animals that underwent surgicalgastric bypass and maintained on high fat diet (RYGB), animals thatunderwent sham surgery and maintained on high fat diet (SHAM), andanimals that underwent sham surgery and were weight matched to the RYGBanimals (WMS). See FIG. 2.

Animals were weighed weekly and their weights were compared topre-operative weights. FIG. 3A shows the percent change of thepre-operative weights for the three groups of mice every week until 10weeks post-surgery. The weights in the SHAM group steadily increased togreater than pre-surgery weights. RYGB group and WMS group significantlylowered weights post-surgery. The RYGB group decreased to approximately70% of pre-surgery weights after 2 weeks, whereas the WMS group steadilydecreased until 10 weeks post-surgery. Complete weight-matching occurredand was stabilized by 6 weeks post-surgery (3 weeks after foodrestriction).

TABLE 1 One-week food intake and fecal calorimetry analysis. Valuesrepresented as means ± SEM. Data not annotated by the same letter aresignificantly different (P < 0.05 ANOVA). RYGB SHAM WMS N 14 11 6 Totaloutput (g)  5.2 ± 0.37^(A)  2.7 ± 0.08^(B)  2.2 ± 0.13^(B) Fecal kcal/g 5.4 ± 0.11^(A)  3.4 ± 0.04^(B)  3.4 ± 0.11^(B) Fecal Fat % 24.5 ±2.3^(A)   0.5 ± 0.09^(B)  1.0 ± 0.52^(B) Fecal Nitrogen %  2.3 ±0.14^(A)  2.6 ± 0.21^(A)  2.3 ± 0.32^(A) Fecal pH  7.0 ± 0.09^(A)  8.0 ±0.04^(B)  8.3 ± 0.20^(B) Food intake (kcals) 121.9 ± 4.2^(A)  114.1 ±2.1^(A)  86.92 ± 1.6^(B)  Fecal energy (kcals) 28.6 ± 2.4^(A)  9.1 ±0.3^(B) 7.7 ± 0.5^(B) Net Energy_(in) (kcals) 93.2 ± 2.5^(A)  105.0 ±1.8^(B)  79.3 ± 1.3^(C)  Fat intake (g) 8.14 ± 0.28^(A) 7.63 ± 0.14^(A)5.81 ± 0.11^(B) Fecal Fat (g) 1.36 ± 0.19^(A) 0.02 ± 0.01^(B) 0.03 ±0.01^(B) Net Fat_(in) (g) 6.78 ± 0.16^(A) 7.61 ± 0.14^(B) 5.78 ±0.10^(A) N intake (g) 6.05 ± 0.21^(A) 5.67 ± 0.10^(A) 4.31 ± 0.08^(B)Fecal N (g) 0.12 ± 0.01^(A)  0.07 ± 0.004^(B) 0.05 ± 0.01^(B) NetNitrogen_(in) (g) 5.92 ± 0.20^(A) 5.59 ± 0.10^(A) 4.26 ± 0.08^(B)

As shown in Table 1, the RYGB group had a net energy intake lower thanthe SHAM group and higher than the WMS group.

FIG. 3B shows food intake of the three groups. Asterisks are used toidentify statistically significant differences. Food intake in the RYGBgroup was not significantly different from the SHAM group, whereas theWMS group was fed ˜25% less food in order to match the weight of theRYGB group. The RYGB group lost significantly more energy in their fecesthan both the SHAM group and the WMS group. See FIG. 3C. FIG. 3D showsthe net energy intake for each group, which was calculated as thedifference between the food intake and the fecal energy output. The netenergy intake of the RYGB group was significantly lower than the netenergy intake of the SHAM group and significantly higher than the netenergy intake of the WMS group. See FIG. 3D.

As shown in Table 2, RYGB mice maintained on a high fat diet exhibitedlower plasma glucose and insulin levels at 15 weeks post-surgery, ascompared to SHAM animals, and similar levels to those of low fat dietanimals. There is a significant improvement in insulin sensitivity inthe RYGB group relative to the SHAM group, as indicated by homeostaticmodel assessment for insulin resistance (HOMA-IR).

TABLE 2 2-hour Fasted Blood Concentrations. Values are represented asmeans ± SEM. Data not annotated by the same letter are significantlydifferent (P < 0.05, ANOVA). RYGB SHAM WMS DIO LF N 17 6 6 8 3 Glucose 137 ± 6.9^(A)  172 ± 8.7^(B)  143 ± 8.8^(A)  209 ± 11.1 122 ± 2.0 (mg/dL) Insulin 2.18 ± 0.39^(A) 5.28 ± 2.0^(B) 0.35 ± 0.05^(C) 25.0 ±5.10 2.31 ± 0.89 (ng/mL) HOMA-IR 18.3 ± 3.2^(A) 57.2 ± 21.6^(B)  3.0 ±0.48^(C) 317.7 ± 64.8  17.4 ± 6.7  Tg (mg/dL) 35.4 ± 7.1^(A) 75.4 ±35.3^(A) 20.9 ± 2.4^(A) 25.0 ± 2.2  63.7 ± 19.3 NEFA 0.18 ± 0.02^(A)0.19 ± 0.03^(A) 0.11 ± 0.02^(B) 0.23 ± 0.01 0.15 ± 0.03

Glucose tolerance and insulin tolerance were measured from the threegroups of mice at 15 weeks post-surgery. FIG. 3E shows an improvement inoral glucose tolerance between the RYGB and WMS groups versus the SHAMgroup. FIG. 3F shows a significant improvement in insulin sensitivity inthe RYGB group as compared to the SHAM and WMS groups.

Triglyceride levels were measured in both liver and serum for animals ineach group. SHAM animals had higher triglycerides in both the liver(FIG. 4) and serum (FIG. 5) than RYGB and WMS animals.

Body composition was determined from whole body lean and fat mass fromthe three groups of mice post mortem at 12-15 weeks after surgery. FIG.6 shows the RYGB group had significantly less fat mass than the SHAMgroup. The RYGB group also has greater lean mass than both WMS and SHAMgroups.

In addition, the adiposity index was determined from epididymal andretroperitoneal fat pads collected from animals of the three groups.Both RYGB and WMS groups had significantly lower percentage of adipositythan the SHAM group. See FIG. 7.

Example 3 Donor Microbiota Profiles

Fecal samples were collected post-surgery from animals in the threegroups every week to measure temporal effects of gastric bypass onoverall microbial diversity. FIG. 8 diagrams the protocol that was usedfor analysis of the fecal samples.

The microbial diversity was compared in samples of isolated bacterialDNA obtained from contents of the gastric stomach, distal stomachremnant, BP limb, Rx limb, common limb (first 3 cm distal to BP/Rxanastomosis), the distal ileum, ½ cecum, and colon. Bacterial DNA fromthe samples was analyzed by an unweighted UniFrac analysis of theprincipal coordinate (PC) for surgery. FIG. 9 shows the UniFrac-basedanalysis showing the change in microbial populations in animals beforeand after RYGB or sham operation. Clustering of the sample coordinatesindicates similar microbial ecologies prior to surgery, with a markedshift in the RYGB communities within a week after surgery that continuedover time and stabilized after 5 weeks.

Interestingly, the sham surgery had a small but equivalent effect in thefecal microbial populations, independent of food restriction.Furthermore, the differences in microbial ecology between SHAM and WMSgroups were minimal. Samples from donors that received gastric bypassmaintained on high fat diet (RYGB) and animals that received gastricbypass maintained on low fat diet (LF-RYGB) clustered together,suggesting that the effect of surgery on shaping the gut microbialcommunities is independent of diet. See FIG. 9.

UniFrac population comparison is shown in FIG. 10 of the three treatmentgroups for each of the locations from which microbial contents werecollected: gastric pouch, distal stomach remnant, BP limb, Rx limb,common limb (first 3 cm distal to BP/Rx anastomosis), the distal ileum,½ cecum, and colon indicating the location along the length of theintestinal tract at which the greatest changes in microbial ecologyoccur (distal segments: ileum, cecum, colon; and stomach remnant; bothluminal content and mucosal adherent populations) after RYGB. There isalso evidence of changes in the microbial communities of mucosaladherent colonies along the entirety of the small intestine (BP, Roux,common limb, and ileum) in WMS animals. The linear discriminant analysis(LDA) effect size (LEfSe) method was used to identify bacterial taxa andspecies-level phenotypes whose relative abundance varied significantlyamong fecal samples taken from the RYGB, SHAM, and WMS groups. An LDAscore >2 shows significant variation. As shown in Table 3, there was asignificant increase in Alistipes and Akkermansia after RYGB throughoutthe gastrointestinal tract.

TABLE 3 Taxonomic Groups with Differential Relative Abundance BetweenTreatments Taxonomicgroup Association LDA score p_Bacteroidetes RYGB2.65 p_...      c_Bacteroidia RYGB 2.65p_....     c_....    o_Bacteroidales RYGB 2.65p_....     c_....    o_....    f_Rikenellaceae RYGB 2.10p_....     c_....    o_....    f_....    g_Alistipes RYGB 2.10p_....     c_....    o_....    f_....    g_....  s_AlistipesfinegoldiiRYGB 2.10p_....     c_....    .o_....     f_....    g_....  s_Alistipesfinegoldii.109956RYGB 2.10 p_Firmicutes.c_Clostridia.o_Clostridiales.f_ClostridiaceaeRYGB 2.42 p_....    c_....    o_....    f_....     g_Clostridium RYGB2.42p_....    c_....    o_....    f_....     g_....    s_ClostridiumperfringensRYGB 2.34p_....    c_....    o_....    f_....     g_....    s_Clostridiumperfringens.46789RYGB 2.34 p_Proteobacteria RYGB 2.37 p_....      c_GammaproteobacteriaRYGB 2.34 p_....      c_....      o_Enterobacteriales RYGB 2.34p_....      c_....      o_....      f_Enterobacteriaceae RYGB 2.34p_....      c_....      o_....      f_....      g_Escherichia RYGB 2.13p_....      c_....      o_....      f_....      g_....     s_(—) RYGB2.13p_....      c_....      o_....      f_....      g_....     .s_.169182RYGB 2.13 p_Verrucomicrobia RYGB 2.27 p_....       c_VerrucomicrobiaeRYGB 2.27 p_....       c_....      o_Verrucomicrobiales RYGB 2.27p_....       c_....      o_....      f_Verrucomicrobiaceae RYGB 2.27p_....       c_....      o_....      f_....      g_Akkermansia RYGB 2.27p_....       c_....      o_....      f_....      g_....    s_(—) RYGB2.27p_....       c_....      o_....      f_....      g_....    s_.178399RYGB 2.27 p_Firmicutes SHAM 2.77 p_....    c_Bacilli SHAM 2.22p_....    c_....  o_Lactobacillales SHAM 2.22p_....    c_....  o_....     f_Lactobacillaceae SHAM 2.17p_....    c_....  o_....     f_....     g_Lactobacillus SHAM 2.17p_Tenericutes SHAM 2.42 p_....    c_Erysipelotrichi SHAM 2.42p_....    c_....      o_Erysipelotrichales SHAM 2.42p_....    c_....      o_....      f_Erysipelotrichaceae SHAM 2.42p_....    c_....      o_....      f_....      g_Allobaculum SHAM 2.41p_....    c_....      o_....      f_....      g_....   s_AllobaculumsplD4SHAM 2.13p_....    c_....      o_....      f_....      g_....   s_AllobaculumsplD4.115098SHAM 2.09 p_Bacteroidetes.c_Bacteroidia.o_Bacteroidales.f WMS 2.54p_....     c_....    o_....    f_.... g WMS 2.54p_....     c_....    o_....    f_.... g_.... s WMS 2.54p_....     c_....    o_....    f_.... g_.... s_.442151 WMS 2.31p_Firmicutes.c_Clostridia WMS 2.69 p_....    c_....    o_ClostridialesWMS 2.69 p_....    c_....    o_....    f_Lachnospiraceae WMS 2.44p_....    c_....    o_....    f_Lachnospiraceae.g_(—) WMS 2.42p_....    c_....    o_....    f_....     g_.... s_(—) WMS 2.42p_Firmicutes.c_Clostridia.o_Clostridiales.f_Lachnospiraceae.g_.s_.190063WMS 2.01 p_....    c_....    o_....    f_Ruminococcaceae WMS 2.45p_....    c_....    o_....    f_....    g_(—) WMS 2.40p_....    c_........   o_........   f_........   g_.... s_(—) WMS 2.40

FIG. 11A shows one taxon that demonstrated changes in microbialdiversity after RYGB surgery. A percentage change of Enterobacterialeswas seen in fecal samples of post surgery RYGB animals. In fact, thepercentage of Enterobacteriales taxon present prior to surgery wasextremely low in all animals. FIG. 11B shows the relative abundance ofEnterobacteriales before and during the 12 weeks following surgery infecal samples collected from RYGB, SHAM and WMS animals. The relativeabundance of Enterobacteriales in RYGB animals increased post surgeryand the increased levels were sustained. FIG. 11C shows the relativeabundance of luminal contents and mucosa across the length of thegastrointestinal tract, comparing RYGB animals to SHAM and to WMScontrols.

FIG. 12 further shows the average relative abundance of bacterial ordersin RYGB, SHAM, and WMS mice in fecal samples collected before surgeryand every week thereafter until 12 weeks post-surgery. Interestingly,RYGB demonstrated a unique increase and decrease in the prevalence ofmultiple microbial populations not seen after diet-induced weight lossalone, including an increase in Enterobacteriales, Verrucomicrobiales,and Bacteroidales populations and a decrease in Clostridiales,Lactobacillales, and Erysipelotrichales of the Fimicutes phylum. Theincreased prevalence of Enterobacteriales, Verrucomicrobiales, andBacteroidales populations and a decreased abundance of Clostridiales,Lactobacillales, and Erysipelotrichales are also seen in FIG. 13 wherethe average relative abundance of bacterial orders is analyzed fromcontents collected from the different gastrointestinal regions: gastricpouch, distal stomach remnant, BP limb, Rx limb, common limb (first 3 cmdistal to BP/Rx anastomosis), the distal ileum, ½ cecum, and colon. TheWMS mice only exhibited increases in Verrucomicrobiales (e.g.,Akkermansia) in the mucosal layer, whereas the RYGB mice exhibitedincreases throughout. See FIG. 13.

FIG. 14 shows a cladogram depicting the hierarchical structure ofrelative abundance of microbial taxa present in the fecal samples ofRYGB, SHAM, and WMS mice. Prevalence of Bacteroidetes, Verrucomicrobia,Proteobacteria, and Firmicutes phyla in RYGB mice; Firmicutes andTenericutes in SHAM mice; and Bacteroidetes and Firmicutes in WMS miceis shown. Significant phyla are labeled, followed by the genera inparentheses.

Example 4 Recipient Analysis

Cecal contents from a donor animal of each group, RYGB- andsham-operated (SHAM), were saved and administered by oral gavage togerm-free recipient mice to yield RYGB-R, and SHAM-R mice. Uninnoculatedgerm-free mice (not shown) were also used as controls. See FIG. 15.

Animals were weighed prior to microbiota transfer and days 1, 7, and 13after microbiota transfer, and their weights were compared to pre-gavagetreatment weights. FIG. 16 shows the percent change of the weights forthe three groups of mice over time, pre-gavage to 13 days post-gavage.The weights in the SHAM-R and germ-free groups demonstrated slight, butnon-significant changes from pre-gavage weights. RYGB-R group decreasedweights to approximately 5% weight post-gavage. RYGB-R recipients' finalweights decreased significantly, whereas both the germ-free and SHAM-Rrecipients' final weights were not significantly different from theirpre-gavage weights (FIG. 17).

Recipient animals were housed individually on wire floors to weigh foodintake at day 0, 7, and 13 after colonization. FIG. 18 shows total foodintake during the 2 week colonization of the three groups. Food intakein the RYGB-R group was not significantly different from the germ-freegroup, while the SHAM-R group ate significantly less than the GF group.

TABLE 4 Effect of transferred microbiota on glucose metabolism and fatcontent. Values are represented as means ± SEM. Data not annotated bythe same letter are significantly different (P < 0.05, ANOVA). RYGB-RSHAM-R GF N 15 10 7 Fasted blood glucose (mg/dL) 148 ± 5.5^(A)  147 ±7.4^(A)  120 ± 3.9^(B)  Fasted insulin (ng/mL) 0.46 ± 0.10^(A) 0.99 ±0.19^(A) 0.73 ± 0.19^(A) HOMA-IR  4.4 ± 0.98^(A) 9.8 ± 4.1^(A) 5.5 ±1.5^(A) Fat pad weight (g) 0.86 ± 0.11^(A) 1.49 ± 0.11^(B) 0.96 ±0.11^(A) Fat pad weight  2.5 ± 0.30^(A)  3.9 ± 0.27^(B)  2.8 ± 0.32^(A)(% body weight)

As shown in Table 4, the recipients of microbiota from RYGB donors haddecreased fat pad weight. The adiposity index was determined fromepididymal and retroperitoneal fat pads collected from the recipientanimals of the three groups. Both germ-free and RYGB-R recipients hadsignificantly lower percentage of adiposity than the SHAM-R group. SeeFIG. 19. In addition, there is a trend towards improvement in insulinsensitivity in the RYGB-R group relative to the SHAM-R group, asindicated by homeostatic model assessment for insulin resistance(HOMA-IR). Plasma leptin levels taken at end of study from recipientanimals colonized with cecal contents from RYGB or SHAM operated donorsare shown in FIG. 20. Leptin levels appear to be correlated withadiposity index of recipient animals.

Serum triglyceride levels were measured in serum from recipients of eachgroup. Like the donor animals shown in FIG. 5, SHAM-R animals had highertriglycerides than RYGB-R and germ-free animals. See FIG. 21.

Energy expenditure was measured via doubly labeled water in recipientsof each group. RYGB-R animals exhibited a non-significant trend towardshigher energy expenditure than SHAM-R and germ-free animals. See FIGS.22A and 22B. The respiratory quotient (RQ), or respiratory exchangeratio, was calculated as VCO₂/VO₂ as an indicator of substrate oxidationfor fuel. FIGS. 23A and 23B show an overall lower RQ value in recipientanimals two weeks following colonization with RYGB microbiota ascompared with RQ values of recipient animals colonized with SHAMmicrobiota. FIG. 23C shows that the RQ differences are significantduring both light and dark periods, with a greater fold drop in RQ inthe light period. Together, FIGS. 23A-C demonstrate a preference forlipid oxidation in RYGB-R animals as compared with SHAM-R animals,particularly during the light phase of the daily light-dark cycles.

Cecal short chain fatty acid (SCFA) composition was measured in bothdonor and recipient animals. See FIGS. 24A-D. Total cecal SCFAs (FIGS.24A and 24C) and percentage of total SCFAs that were acetate,propionate, and butyrate (FIGS. 24B and 24D) were measured. See FIGS.24A-D. The relative proportion of acetate, propionate and butyratelevels were maintained in both donor and recipient groups with increasedpropionate production and decreased acetate production. In the recipientgroup, SHAM-R animals produced significantly more SCFAs than germ-freeanimals, and RYGB-R produced an intermediate quantity. The difference isnot as apparent in the donor group.

Example 5 Recipient Microbiota Profiles

Fecal samples were collected post-gavage from recipient animals of theRYGB cecal content, SHAM cecal content or WMS cecal content at intervalsof 1, 2, 3, 7, and 13 days to determine the sustainability of themicrobial diversity from the transferred cecal contents within theun-operated, normal gastrointestinal anatomy of recipient animals.

The microbial diversity was compared in samples of isolated bacterialDNA of fecal samples collected from RYGB-R, SHAM-R and WMS-R recipientanimals. FIG. 25 shows the relative abundance of bacterial orders.Unlike the increase in Enterobacteriales in the RYGB animals in FIG. 12,the RYGB-R experimental groups demonstrated a reduced abundance of thisparticular order of bacteria by the end of the colonization period.Interestingly, the increased abundance of the Verrucomicrobiales groupis sustained throughout the colonization period, which is uniquecompared to the SHAM-R and WMS-R samples. Table 5 shows an overallsignificant increase in Alistipes and Akkermansia throughout thegastrointestinal tract of the RYGB-R group.

TABLE 5 Taxonomic Groups with Differential Relative Abundance BetweenRecipients Taxonomic group Association LDA score p  Bacteroidetes.c Bacteroidia.o  Bacteroidales.f RYGB 2.87p_.....     c_.....    o_.....    f_.....    g RYGB 2.87p_.....     c_.....    o_.....    f_.....    g_..... RYGB 2.87p_.....     c_.....    o_.....    f_Rikenellaceae RYGB 2.39p_.....     c_.....    o_.....    f_.....    g_Alistipes RYGB 2.39p_.....     c_.....    o_.....    f_.....    g_.....   s_AlistipesfinegoldiiRYGB 2.42p_.....     c_.....    o_.....    f_.....    g_.....   s_Alistipesfinegoldii.109956RYGB 2.42 p_Firmicutes.c_Clostridia.o_Clostridiales.f_ClostridiaceaeRYGB 2.23 p_.....    c_.....    o_.....    f_.....    g_Clostridium RYGB2.22 p_Proteobacteria RYGB 2.74 p_.....     c_Gammaproteobacteria RYGB2.74 p_.....     c_.....    o_Enterobacteriales RYGB 2.67p_.....     c_.....    o_.....     f_Enterobacteriaceae RYGB 2.67p_Verrucomicrobia RYGB 3.03 p_.....     c_Verrucomicrobiae RYGB 3.03p_.....     c_.....    o_Verrucomicrobiales RYGB 3.03p_.....     c_.....    o_.....    f_Verrucomicrobiaceae RYGB 3.03p_.....     c_.....    o_.....    f_.....      g_Akkermansia RYGB 3.03p_.....     c_.....    o_.....    f_.....      g_.....    s RYGB 3.03p_.....     c_.....    o_.....    f_.....      g_.....    s_.178399 RYGB3.03p_Bacteroidetes.c_Bacteroidia.o_Bacteroidales.f_.....   g_.....     s_.234036SHAM 3.04 p_.....    c_.....    o_.....    f_Porphyromonadaceae SHAM2.98 p_.....    c_.....    o_.....    f_.....    g_Parabacteroides SHAM2.98 p_.....    c_.....    o_.....    f_.....    g_.....     s SHAM 2.98p_.....    c_.....    o_.....    f_.....    g_.....     s_.249661 SHAM2.98 p_Firmicutes SHAM 3.11 p_.....    c_Bacilli SHAM 2.73p_.....    c_.....   o_Lactobacillales SHAM 2.73p_.....    c_.....   o_.....    f_Lactobacillaceae SHAM 2.25p_.....    c_.....   o_.....    f_.....    g_Lactobacillus SHAM 2.25p_.....    c_.....   o_.....    f_.....    g_.....    s SHAM 2.25p_Firmicutes.c_Bacilli.o_Lactobacillales.f_Lactobacillaceae.g_Lactobacillus.s_.323257SHAM 2.25 p_Firmicutes.c_Clostridia SHAM 2.91p_.....    c_.....    o_Clostridiales SHAM 2.91p_.....    c_.....    o_.....    f_Lachnospiraceae SHAM 2.85p_.....    c_.....    o_.....    f_.....    g SHAM 2.83p_.....    c_.....    o_.....    f_.....    g_.....    .s SHAM 2.83p_.....    c_.....    o_.....    f_.....    g_.....    s_.136452 SHAM2.85p_Proteobacteria.c_Gammaproteobacteria.o_Enterobacteriales.f_Enterobacteriaceae.g_ErwiniaSHAM 2.39 p_.....    c_.....    o_.....    f_.....    g_.....    s SHAM2.39 p_.....    c_.....    o_.....    f_.....    g_.....    s_.9822 SHAM2.39p_Bacteroidetes.c_Bacteroidia.o_Bacteroidales.f_.....    g_.....    .s_.183770WMS 2.49p_.....    c_.....    o_.....    f_.....    g_.....    s_.206324 WMS2.03 p_.....    c_.....    o_.....    f_.....    g_.....    s_.442151WMS 2.65 p_.....    c_.....    o_.....    f_Bacteroidaceae WMS 3.23p_.....    c_.....    o_.....    f_.....    g_Bacteroides WMS 3.23p_.....    c_.....    o_.....    f_.....    g_.....    s WMS 3.23p_.....    c_.....    o_.....    f_.....    g_.....    s_.348374 WMS3.19 p_.....    c_.....    o_.....    f_.....    g_.....    s_.86458 WMS2.26p_Proteobacteria.c_Gammaproteobactena.o_Enterobacteriales.f_Enterobacteriaceae.g_EscherichiaWMS 2.68 p_.....    c_.....    o_.....    f_.....    g_.....    s WMS2.68 p_.....    c_.....    o_.....    f_.....    g_.....    s_.169182WMS 2.68 p_Tenericutes WMS 2.26 p_.....   c_Erysipelotrichi WMS 2.26p_.....   c_.....      o_Erysipelotrichales WMS 2.26p_.....   c_.....      o_.....    f_Erysipelotrichaceae WMS 2.26p_.....   c_.....      o_.....    f_.....    g_Allobaculum WMS 2.28p_.....   c_.....      o_.....    f_.....    g_.....    s WMS 2.28p_.....   c_.....      o_.....    f_.....    g_.....    s_.227728 WMS2.28

What is claimed is: 1.-13. (canceled)
 14. A pharmaceutical compositionfor altering microbiota comprising: a therapeutically effective amountof at least one of a substantially purified Verrucomicrobia,substantially purified Bacteroidetes, substantially purified Firmicutesand substantially purified Proteobacteria; and a pharmaceuticallyacceptable carrier.
 15. The composition of claim 14 wherein thesubstantially purified Verrucomicrobia is Verrucomicrobiales, thesubstantially purified Bacteroidetes is Bacteroidales, the substantiallypurified Firmicutes is Clostridiales and the substantially purifiedProteobacteria is Enterobacteriales.
 16. The composition of claim 14wherein the substantially purified Verrucomicrobia is Akkermansia, thesubstantially purified Bacteroidetes is Alistipes, the substantiallypurified Firmicutes is Clostridium and the substantially purifiedProteobacteria is Escherichia.
 17. The composition of claim 14formulated for oral delivery.
 18. The composition of claim 14 furthercomprising a therapeutically effective amount of a compound to alter atleast one of Bacteroidetes, Verrucomicrobia, Firmicutes, Tenericutes,and Proteobacteria. 19.-27. (canceled)
 28. A kit for measuring therelative abundance of at least one of Verrucomicrobia, Proteobacteria,Bacteroidetes, Tenericutes, and Firmicutes in a sample comprising: apair of primers that specifically hybridize to Verrucomicrobia,Proteobacteria, Bacteroidetes, Tenericutes or Firmicutes nucleic acids;and hybridization reagents. 29.-35. (canceled)
 36. A compositioncomprising one or more of: a) substantially purified Verrucomicrobia; b)substantially purified Proteobacteria; c) substantially purifiedBacteroidetes; d) substantially purified Firmicutes; e) substantiallypurified Alistipes; f) substantially purified Escherichia; g)substantially purified Clostridium; h) substantially purifiedAkkermansia and i) a compound that when administered to a subjectincreases a relative abundance of at least one of Verrucomicrobia,Bacteroidetes, Firmicutes and Proteobacteria in the subject and/ordecreases a relative abundance of Tenericutes and Erysipelotrichales inthe subject; and optionally comprising one or more of: i) a compoundthat when administered to a subject alters a relative abundance of atleast one of Bacteroidetes, Verrucomicrobia, Firmicutes, Tenericutes andProteobacteria; j) substantially purified Bacteroidetes; and k)substantially purified Proteobacteria. 37.-44. (canceled)